Zirconia toughened alumina (ZTA) powders: ultrastructural and histological analysis.

Ceramic materials, as Alumina and Zirconia, has made an improvement in the choice of new biomaterials for the load bearing application in dental and orthopaedic implants. These materials has shown mechanical resistance to high stress related to weight bearing and low debris in time. For this reason they are indicated on young patients implant, with high demanding activities and long life expectance. In literature however the risk of chronic inflammation due to chronic wear debris release and the possibility of carcinogenesis, is still to be definitively investigated. Another point to investigate is the acute reaction of the tissue in case of acute release of powders of these materials. The aim of this study was to investigate the possible local and systemic acute effects of ceramic precursors in form of powders of different size when released into articular joint. Powders of ZTA were implanted in the knee joint of twenty-four New Zealand white adult rabbits, that were sacrificed at 1,3,6, and 12 months. Radiographic, histological and immunoistochemestry analysis were conducted on periprosthetic tissue and peripheral organs, to verifying local host response and systemic toxic effects.

In vivo characterization of Zirconia Toughened Alumina material: a comparative animal study.

The development of a new chromia-doped Zirconia Toughened Alumina (ZTA) material was previously reported as displaying mechanical properties suitable for implants with load bearing applications, such as orthopaedic and dental implants. This type of biomaterial is expected to be in contact with living tissues for a long period of time and its long-term toxicity must be carefully evaluated. In this study the suitability of this ZTA material as a candidate biomaterial for orthopaedic implants and dental devices was further investigated in vivo in comparison to alumina and zirconia, which are currently used in orthopaedic and dental surgery. Cylinders of the materials were implanted in vivo in white rabbits, and local and systemic tissue reactions were analyzed at different time intervals after surgery. Radiologic examinations displayed the absence of radiolucence around cylinders and no signs of implant loosening up to twelve months. No tumours developed in the animals either locally (at the site of implantation), or systemically in the peripheral organs. The results obtained suggest that this new ZTA material does not display any long term pathogenic effect in vivo. These findings extend our previous observations on the biocompatibility and the absence of any long-term carcinogenic effect in vitro of this material which displays interesting properties for biomedical applications. In conclusion, we report the in vivo characterization of a new chromia-doped ZTA material and confirm its suitability as a candidate biomaterial for orthopaedic implants and dental devices since it does not give any local nor systemic toxicity even after a long period of time after implantation.

The cutaneous microvascular architecture of human diabetic toe studied by corrosion casting and scanning electron microscopy analysis.

In this morphological study, we report on the three-dimensional microvascular architecture constituting the toes of a patient affected by diabetic microangiopathy. We applied corrosion casting (CC) technique to the toes of a patient affected by Type 2 diabetes, who underwent surgery for explantation of inferior left limb due to necrotic processes of soft tissues. The toes of a foot traumatically explanted in a motorcycle accident were kept as controls. According to technical protocols, toes were injected with a low-viscosity acrylic resin (Mercox) through the major digital artery, tissues were corroded in KOH solution (8%), and resulting casts processed for SEM observations. Already at low magnification, in diabetic toes, we found an impairment of the linear track-like disposition of the vessels of plantar side, with signs of vascular disruption and obliterations, stopped resin, and leakages. Capillaries under the nail and a lot of vascular villi in eponychium and nail borders were damaged, and vascular regression phenomena acting on them were clearly visible. Resin leakages and impairment of normal vascular architecture were also observed in the root of the nail. This preliminary report represents only the first step for further investigations regarding morphological three-dimensional appearance of diabetic microangiopathy. CC and scanning electron microscopy technique well documented these morphological modifications, highlighting on both structural and ultrastructural features of diabetic toes microvessels. In conclusion, our qualitative data try to better focus on the pathophysiological mechanisms involved in diabetic dermopathy and microangiopathy, proposing CC as useful method to investigate on them.

Development of a new zirconia-toughened alumina: promising mechanical properties and absence of in vitro carcinogenicity.

High purity alumina as well as zirconia ceramics have been widely used as orthopaedic implant biomaterials and dental devices displaying optimal, but sometimes exclusive, mechanical properties. In order to combine the advantages of alumina and zirconia ceramic materials different types of composites have been developed in which either zirconia is dispersed in an alumina matrix or vice versa. Orthopaedic and dental implant biomaterials are expected to be in contact with living tissues for a long period of time and their long term toxicity must be carefully evaluated. In this study we report the development of a high performance chromia-doped zirconia toughened alumina (ZTA) material which displays promising mechanical properties in terms of hardness, strength and fracture toughness that make it suitable for prosthesis even for small joints. The long-term biocompatibility of this material was also evaluated, mainly in terms of DNA damage, mutagenicity and cancerogenetic potential in mammalian cells. The results obtained suggest that this new ZTA material does not display any longterm carcinogenic effect and it is suitable for biomedical applications from a cancerogenetic point of view. In conclusion, we report the development of a new chromia-doped ZTA material with interesting properties, both from a mechanical and a biocompatibility point of view which warrant further studies on its suitability as a candidate biomaterial for orthopaedic implants and dental devices.

Relationship between intrathecal baclofen and the central nervous system.

The GABA(B) receptor agonists display a number of pharmacological effects including central muscle relaxation, decreased self-administration of cocaine and narcotic drugs, antinociception, cognitive impairment as well as enhancement of synaptic plasticity. The main relationships between intrathecal or intracerebral baclofen and the Central Nervous System (CNS) are reviewed with particular attention to actions on pain, epilepsy and basal ganglia regulation. Since baclofen may be involved in synaptic plasticity and the development of neuronal pathways, the main issues of this field are reviewed with particular attention to the effects of baclofen on the developing brain. The role of baclofen in the regulation of movement has not been clearly understood, but recent findings support its important involvement in globus pallidus and subthalamic nucleus. The neuroprotective action of baclofen in cerebral ischemia is a matter of debate. The effects of baclofen in cognition and attention are another important issue because patients with chronic intrathecal baclofen (ITB) administration often present with impairment of cognitive functions. Drug craving and its improvement after baclofen administration is also reviewed. Finally, the clinically interesting results on the regulation of food intake and blood pressure are highlighted. The preliminary experience on the effects in cortical neuron viability at different concentrations of ITB is reported.

3D analysis of SEM images of corrosion casting using adaptive stereo matching.

The corrosion casting method represents one of the most widely used technique to study the 3D microvascularization of many tissues, both in their normal and pathological conditions. For a long time this technique was used only to perform a qualitative evaluation of the images obtained by scanning electron microscopy (SEM). A quantitative evaluation of vascular parameters (e.g., interbranching and intervascular distances, angle measurements, lengths and diameters) was lacking, mainly because of the difficulties found in the measurement performed on 2D SEM images. Then, some authors reported a quantitative method based on the analyses of stereo-pair images that allowed precise morphometric measurements. To visualize the specimens in 3D, it was necessary to use red-green glasses. In this article we describe a new approach by which we can automatically obtain a 3D reconstruction of vascular cast specimen's surface directly from stereo-images. Moreover, we developed a software that performed micrometric measurements on the 3D construct generated from the stereo-pictures. In conclusion, implementing together these two softwares and applying them to corrosion casting samples made it possible to render in 3D the surface of corrosion cast as well as make quantitative measurements on the corrosion casts.

The three-dimensional microvascular architecture of the human Kaposi sarcoma implanted in nude mice: a SEM corrosion casting study.

The human Kaposi sarcoma represents one of the most common skin lesions associated with AIDS. Its clinical presentation and anatomopathological structure seem to demonstrate a particularly rich vascularity. The latest therapies aim to limit its intrinsic angiogenic activity in an attempt to reduce vascular density and the formation of new vessels. For these reasons, we decided to study the microvascular architecture of Kaposi sarcoma in three dimensions. We used a corrosion casting technique applied to nude mice previously transplanted subcutaneously with human modified neoplastic Kaposi sarcoma cells. The cooption of host vessels made by the tumor was demonstrated by three-dimensional scanning electron microscopy (SEM) images. At high magnification several angiogenic patterns were observed in the form of potato-shaped vessels, sprouts, intussusceptions and mouse tailed end tipped capillaries along with some ultrastructural features such as intercellular extravasations and endothelial cell modifications. Our investigation allowed us to build a detailed map of tumor vasculature in human Kaposi sarcoma. Furthermore, this study want to shed light on the sharp morphological three-dimensional conformation of angiogenic sprouts so to be able to better understand their modifications occurred during time and after antiangiogenic experimental therapies, by now observed only by immunohistochemical or immunofluorescent assays.

Plexiform vascular structures in the human digital dermal layer: a SEM--corrosion casting morphological study.

This study aimed to describe the impressive diversity of vascular plexiform structures of the hypodermal layer of human skin. We chose the human body site with the highest concentration of dermal corpuscles, the human digit, and processed it with the corrosion casting technique and scanning electron microscopy analysis (SEM). This approach proved to be the best tool to study these microvascular architectures, free from any interference by surrounding tissues. We took high-definition pictures of the vascular network of sweat glands, thermoreceptorial and tactile corpuscles, the vessels constituting the glomic bodies and those feeding the hair follicles. We observed that the three-dimensional disposition of these vessels strictly depends on the shape of the corpuscles supplied. We could see the tubular vascularization of the excretory duct of sweat glands and the ovoid one feeding their bodies, sometimes made up of two lobes. In some cases, knowledge of these morphological data regarding the normal disposition in space and intrinsic vascularization structure of the dermal corpuscles can help to explain many of the physiopathological changes occurring during chronic microangiopathic diseases.

Microvascularization of the human digit as studied by corrosion casting.

The aim of this study was to describe microcirculation in the human digit, focusing on the vascular patterns of its cutaneous and subcutaneous areas. We injected a functional supranumerary human thumb (Wassel type IV) with a low-viscosity acrylic resin through its digital artery. The tissues around the vessels were then digested in hot alkali and the resulting casts treated for scanning electron microscopy. We concentrated on six different areas: the palmar and dorsal side of the skin, the eponychium, the perionychium, the nail bed and the nail root. On the palmar side, many vascular villi were evident: these capillaries followed the arrangement of the fingerprint lines, whereas on the dorsal side they were scattered irregularly inside the dermal papillae. In the hypodermal layer of the palmar area, vascular supports of sweat glands and many arteriovenous anastomoses were visible, along with glomerular-shaped vessels involved in thermic regulation and tactile function. In the eponychium and perionychium, the vascular villi followed the direction of nail growth. In the face of the eponychium in contact with the nail, a wide-mesh net of capillaries was evident. In the nail bed, the vessels were arranged in many longitudinal trabeculae parallel to the major axis of the digit. In the root of the nail, we found many columnar vessels characterized by multiple angiogenic buttons on their surface.

The microvasculature of the lateral choroid plexus in the rat: a scanning electron microscopy study of vascular corrosion casts.

The three-dimensional microvascular structure of many organs can be adequately investigated only using the corrosion casting technique. We applied this method, consisting of an injection of low viscosity acrylic resin through the major vessels and the subsequent digestion of the organic component with strong alkali or acids, to the choroid plexus of the lateral ventricles in the rat, focusing on its structural vascular features. This approach allowed a qualitative morphological description of the choroid plexus of the lateral ventricles, revealing several aspects of their capillary architecture as well as the morphological details underlying its main functional activity, essential to cerebrospinal fluid turnover. Observation of the casts with scanning electron microscopy gave a detailed picture of the choroid plexus of the lateral ventricles of the rat and enabled us to distinguish four different regions, depending on the site that the lateral ventricles occupied: the anterior olfactory region, the main central region, the longer branch and the inferior horn. Each region mostly consisted of spiral capillaries and had specific characteristics. At high magnification, the casts revealed distinctive vascular specializations, such as numerous bulges regularly placed on the capillaries. This morphological investigation underpins a better comprehension of the pathological mechanisms involving the choroid plexus in the lateral ventricles.

A new method to make vascular and bronchial casts of voluminous organs.

Vascular and bronchial endocasts represent a useful instrument to study the ramification pattern of these structures. Casts have been made from different materials, such as waxes in ancient times and, more recently, silicon-like compounds or resins (see e.g. Mercox) to study the finest details. These techniques are valuable for small specimens, whereas they are inadequate for very large organs, where technical difficulties require the development of specific instrumentation. In this study we present a new simple injection technique, based on expanded polyurethane, which allows preparing vascular and bronchial trees for macroscopic and microscopic studies. The new injection technique is very easy to carry out, since the propulsion is provided by compressed air, and it does not require special instrumentation. To this aim, endocasts of the entire tracheal-bronchial tree and casts of vascular kidney from different animals were prepared. The specimens have a very low weight, show the finest ramifications, and are very stable and resistant to mechanical stress. To examine microscopically the details of the casts, specimens from the kidney cast were also analyzed by scanning electron microscopy, revealing good preservation of microcirculatory structures, functional sphincters and endothelial cell impressions. Therefore, the technique may be useful for macroscopic studies of large specimens, retaining sufficiently fine details.

Sneddon syndrome, arylsulfatase A pseudodeficiency and impairment of cerebral white matter.

We describe a 11 year-old-boy with Sneddon syndrome, confirmed by skin biopsy, and MR evidence of diffuse cerebral hyperintensity of white matter; he also suffered from pre-perinatal hypoxic-ischemic distress. Arylsulfatase A activity was found reduced because of arylsulfatase A pseudodeficiency. We suggest that the association of pre-perinatal distress, Sneddon syndrome and arylsulfatase A pseudodeficiency is responsible for the diffuse impairment of cerebral white matter, never reported in Sneddon syndrome and similar to described cases of delayed posthypoxic demyelination and arylsulfatase A pseudodeficiency.

Detection of a rare Wilson disease mutation associated with arylsulfatase A pseudodeficiency.

We have studied a patient with Wilson disease (WD), belonging to a family segregating late-onset, dominant cerebellar ataxia. Analysis of the WD gene showed that the patient is a compound heterozygote, carrying the 14His1069Gln mutation from the father and the 8Gly710Ser mutation from the mother. The 8Gly710Ser is a mutation described previously only in a Swedish patient. Our patient is also homozygous for arylsulfatase A pseudodeficiency. This genetic defect, which has been reported in association with other neuropsychiatric syndromes, has not been described in WD.

Leber's hereditary optic neuropathy: biochemical effect of 11778/ND4 and 3460/ND1 mutations and correlation with the mitochondrial genotype.

To clarify the bioenergetic relevance of mtDNA mutations in Leber's hereditary optic neuropathy (LHON), we investigated affected individuals and healthy carriers from six Italian LHON families harboring the 11778/ND4 and the 3460/ND1 mtDNA mutations. The enzymatic activities of mitochondrial complex I and its sensitivity to the potent inhibitors rotenone and rolliniastatin-2 were studied in mitochondrial particles from platelets, in correlation with mtDNA analysis of platelets and leukocytes. In platelets homoplasmic for mutant mtDNA, both 11778/ND4 and 3460/ND1 mutations induced resistance to rotenone and the 3460/ND1 mutation also provoked a marked decrease in the specific activity of complex I. Individuals heteroplasmic in platelets for either mutation showed normal biochemical features, indicating functional complementation of wild-type mtDNA. There was no correlation between the clinical status and mtDNA homo/heteroplasmy in platelets, but the biochemical features correlated with the mitochondrial genotype of platelets. In some cases, the degree of mtDNA heteroplasmy differed in platelets and leukocytes from the same individual with a prevalence of wild-type mtDNA in the platelets. These results imply that biochemical studies on mitochondrial diseases should always be integrated with mtDNA analysis of the same tissue investigated and also suggest that the mtDNA analysis on the leukocyte fraction, as usually performed in LHON, does not necessarily reflect the mutant genotype level of other tissues. The differential tissue heteroplasmy may be more relevant than previously thought in determining disease penetrance.

Familial spasmodic dysphonia with low arylsulphatase A (ASA) level.

Two familial cases of late onset spasmodic dysphonia and low Arylsulphatase A (ASA) are reported. In one case spasmodic dysphonia was associated with negative head tremor and orthostatic tremor, both displayed postural tremor of the upper extremities. A familial predisposition for both focal dystonia and metabolic lysosomal impairment is suggested by similar observations.

Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease associated with point mutations in mitochondrial DNA. The most frequent of these mutations is the G-to-A substitution at nucleotide position 11,778 which changes an evolutionarily conserved arginine with a histidine at position 340 in subunit ND4 of NADH:ubiquinone reductase (respiratory complex I). We report that this amino acid substitution alters the affinity of complex I for the ubiquinone substrate and induces resistance towards its potent inhibitor rotenone in mitochondria of LHON patients. Such changes could reflect a substantial loss in the energy conserving function of NADH:ubiquinone reductase and thus explain the pathological effect of the ND4/11,778 mutation.

Arylsulfatase A pseudodeficiency and Lafora bodies in a patient with progressive myoclonic epilepsy.

Since age 12 years, a 25-year-old woman had a syndrome with myoclonic epilepsy, cerebellar signs, and spontaneous myoclonus. Skin biopsy showed typical Lafora bodies (LB), but she lacked a progressive course and mental impairment, hallmarks of Lafora disease. Lysosomal enzyme assays showed low level arylsulfatase A (ASA) activity. DNA study disclosed a homozygous ASA Pd genotype. Both parents carried one Pd allele. The still-unknown relationship between the pathologic level of ASA activity and myoclonic epilepsies suggests introduction of ASA assays in patients with PME.

Arylsulphatase A (ASA) activity in parkinsonism and symptomatic essential tremor.

Arylsulphatase A (ASA) activity was evaluated in 47 patients with a diagnosis of parkinsonism or essential tremor. Mean ASA activity was significantly reduced compared with both a healthy control group of 71 individuals (p < 0.01) and with a group of 44 neurological patients without movement disorders (p < 0.02). Using definite clinical criteria the patients were classified as typical or atypical with respect to Parkinson's disease (PD) or essential tremor (ET). A normal ASA level was found in all the cases showing typical clinical features (PD and ET), while ASA activity was significantly lowered (p < 0.01) in 55.6% of the atypical cases (Parkinsonian syndrome or symptomatic ET). Our data support the hypothesis of a non-casual association between low ASA level and the clinical features of parkinsonism or symptomatic ET.

Abnormal platelet mitochondrial function in patients affected by migraine with and without aura.

To investigate energy metabolism in migraine, we determined platelet mitochondrial enzyme activities in 40 patients with migraine with aura and in 40 patients with migraine without aura during attack-free intervals and in 24 healthy control subjects. NADH-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities in both patient groups were significantly lower than in controls (p < 0.01), while NADH-cytochrome-c-reductase activity was reduced only in migraine with aura (p < 0.01). No alteration in succinate-dehydrogenase was observed. Monoamine-oxidase activity differed between sexes (p < 0.05) but within each sex group no difference was observed between patients and controls. We hypothesize that the defect in mitochondrial enzymes observed indicates a systemic impairment of mitochondrial function in migraine patients.

Testing models for genetic determination in migraine.

We collected two clinically matched samples of patients, one sample affected by migraine with aura the other by migraine without aura, to investigate the genetic determination of these conditions. A maternal and X-linked transmission for both these diseases was considered unlikely after pedigree analysis. Classical segregation analysis indicated a likely autosomal recessive kind of transmission for both. Reduced penetrance and the h2 values, however, imply the presence of additional genetic and/or environmental factors controlling the phenotypic expression of migraine.