Stefano Squarzoni

Fretting wear in a modular neck hip prosthesis

In vitro cyclic load fretting tests were conducted on a prototype of a cementless, modular neck, hip prosthesis. The study had three major objectives: to determine the amount of fretted material in the tapered-neck joint under various load cycle amplitudes, to determine the fretting damage evolution, and to determine the effect of different-sized stem bodies on the production of debris. All the tests produced some fretting microdamage on the tapered surface although the extent was quite different among test groups. The amount of abraded material increased almost linearly with the applied load magnitude but not with the number of load cycles. The amount of weight loss was higher in the large stem bodies than in the small ones. Weight loss ranged from 0.28 +/- 0.10 mg for small stem bodies loaded 5.5 million times up to 2300N to 2.54 +/- 0.53 mg for large stem bodies located 20 million times up to 3300N. Considering the large-size stem results, and assuming one million load cycles between 300N and 3300N to be the average yearly load history, the modular neck tapered joint would produce 0.6 mg/year of metal debris. The clinical impact of this observation is unknown; however, some of the literature on the presence of metal in patient tissues and fluids supports the hypothesis that a normal and stable prosthesis is likely to produce less than 10 mg/year of metal debris. Thus, a further production of 0.6 mg/year due to the modular neck should not have any significant effect.

Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment

Abstract.Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.

Early diagnosis of ceramic liner fracture. guidelines based on a twelve-year clinical experience

Osteolytic lesions due to wear debris are the major long-term problem associated with total hip replacement1. To avoid wear debris, hard-bearing-surface total hip prostheses with improved tribological properties have been introduced into surgical practice. Ceramic surfaces have had some promising long-term results2, and modern metal-backed alumina cups have been associated with very good clinical results3-5. Alumina has excellent tribological properties and a very high Youngs modulus that leads to very good compression strength, but it has poor bending strength: it has no way to deform6. This means that ceramic can break without warning. Under normal physiologic conditions, modern ceramics never reach their fatigue limit, so ceramic head fractures are rare (a rate of 0.004%7 in one study). In contrast, ceramic liner fractures are not well recognized, and their frequency could be underestimated (Fig. 1). In addition, it is difficult to identify patients who are at risk because liner fractures can be due to multiple causes: dislocation, impingement, malpositioning, and microseparation8,9. Fig. 1 A ceramic liner fracture. The diagnosis is often difficult to make on the basis of standard radiographs. A fragment of ceramic is visible near the calcar (arrow). The liner was found to be fractured (arrow) at revision surgery. While many efforts have been made to improve the ceramic manufacturing process and the surgical technique for inserting ceramic components10, little has been reported regarding the early diagnosis of ceramic fracture. When a ceramic fracture involves the liner and is the consequence of repeated microtrauma, the diagnosis is rarely made early, except when ceramic fragments are visible on radiographs. Moreover, decision-making regarding revision surgery after a ceramic-on-ceramic prosthesis has failed is difficult: the ceramic fragments that have spread into the periarticular space are abrasive and …

Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields

Intramembrane proteins (IMP) represent a class of proteins located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors. Since it has been argued that biological effects of extremely low frequency (ELF) electromagnetic fields are mediated by plasma membrane. this work was designed to study the possible effects of 50 Hz pulsed magnetic fields (PMF) of the type used to stimulate bone repair, on the distribution of IMP in the plasma membrane of Swiss NIH 3T3 fibroblasts. Evaluations were based on the calculation of a distribution factor, which allows discrimination between random, regular and clustered distribution of IMP, in electron microscope images of freeze-fractured membranes. The results indicate that cells exposed to PMF for more than two hours have a significant clustering of the IMP distribution compared to control unexposed cells.

Autosomal recessive myosclerosis myopathy is a collagen VI disorder

Objective: To determine the clinical and molecular features of a new phenotype related to collagen VI myopathies. Methods: We examined two patients belonging to a consanguineous family affected by myosclerosis myopathy, screened for mutations of collagen VI genes, and performed a detailed biochemical and morphologic analysis of the muscle biopsy and cultured fibroblasts. Results: The patients had a novel homozygous nonsense COL6A2 mutation (Q819X); the mutated messenger RNA escaped nonsense-mediated decay and was translated into a truncated α2(VI) chain, lacking the sole C2 domain. The truncated chain associated with the other two chains, giving rise to secreted collagen VI. Monomers containing the truncated chain were assembled into dimers, but tetramers were almost absent; secreted collagen VI was quantitatively reduced and structurally abnormal in cultured fibroblasts. Mutated collagen did not correctly localize in the basement membrane of muscle fibers and was absent in the capillary wall. Ultrastructural analysis of muscle showed an unusual combination of basement membrane thickening and duplication, and increased number of pericytes. Conclusions: This familial case has the characteristic features of myosclerosis myopathy and carries a homozygous COL6A2 mutation responsible for a peculiar pattern of collagen VI defects. Our study demonstrates that myosclerosis myopathy should be considered a collagen VI disorder allelic to Ullrich congenital muscular dystrophy and Bethlem myopathy.

Nuclear changes in a case of X-linked Emery-Dreifuss muscular dystrophy.

Ultrastructural alterations in the nuclear architecture were found in skeletal muscle and skin cultured cells from a patient affected by X‐linked Emery‐Dreifuss muscular dystrophy (EMD) carrying a null mutation. The molecular defect of X‐linked EMD is the absence of emerin, a nuclear envelope‐associated protein which is considered a component of the nuclear lamina. The nuclear changes were present in skeletal muscle and skin cultured cells with a frequency of about 10% and 18%, respectively. The main structures of the nuclear periphery were involved: lamina and nuclear envelope‐associated heterochromatin were affected, whereas the cisterna and the pore complexes appeared preserved, and the cytoplasm of the same cells appeared normal. Analogous localized defects were detectable by immunolabeling with antilamin A/C and B2 antibodies, as well as by selective propidium iodide chromatin staining. The lesions we describe could be the result of anomalous nuclear lamina organization in the absence of emerin.

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: Altered intermolecular interaction with emerin and implications for gene transcription

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription.

Nuclear alterations in autosomal‐dominant Emery‐Dreifuss muscular dystrophy

Electron microscopy study of muscle biopsies from patients with autosomal‐dominant Emery‐Dreifuss muscular dystrophy revealed nuclear alterations in about 10% of the preserved muscle fibers. The major findings consisted of peripheral heterochromatin loss or detachment from the nuclear envelope, and of interchromatin texture alterations. These abnormalities are similar to those reported in an animal model of the disease and to those found in the X‐linked form of Emery‐Dreifuss muscular dystrophy. These results suggest that an abnormal ultrastructural arrangement of the nuclear periphery is a common feature in the known forms of Emery‐Dreifuss muscular dystrophy, and that several proteins of the nuclear scaffold are necessary in muscle cells to maintain the nuclear structural/functional integrity and a normal muscle cell metabolism.

Association of emerin with nuclear and cytoplasmic actin is regulated in differentiating myoblasts.

Emerin is a nuclear envelope protein whose biological function remains to be elucidated. Mutations of emerin gene cause the Emery-Dreifuss muscular dystrophy, a neuromuscular disorder also linked to mutations of lamin A/C. In this paper, we analyze the interaction between emerin and actin in differentiating mouse myoblasts. We demonstrate that emerin and lamin A/C are bound to actin at the late stages of myotube differentiation and in mature muscle. The interaction involves both nuclear alpha and beta actins and cytoplasmic actin. A serine-threonine phosphatase activity markedly increases emerin-actin binding even in cycling myoblasts. This effect is also observed with purified nuclear fractions in pull-down assay. On the other hand, active protein phosphatase 1, a serine-threonine phosphatase known to associate with lamin A/C, inhibits emerin-actin interaction in myotube extracts. These data provide evidence of a modulation of emerin-actin interaction in muscle cells, possibly through differentiation-related stimuli.