Effects of heat deproteinate bone and polynucleotides on bone regeneration: an experimental study on rat.

This experimental study evaluated the effects of polynucleotides on bone regeneration on rats. Defects with a diameter of 2mm were prepared in the thickness of cortical bone of 32 rat tibiae and filled with different compounds: polynucleotide gel (PDRN), deproteinated porcine cortical bone (HDB) obtained by high temperature heating in the form of granules and a paste made of HDB granules and PDRN gel. Bone regeneration of the gaps was histologically analysed after a treatment time ranging from 1 to 12 weeks. Both PDRN and HDB stimulated bone growth and repair, but the paste prepared combining HDB granules and PDRN showed the best performance with faster filling, better osteconductive and biocompatible properties and easier handling. This study suggests that the paste prepared combining HDB and PDRN gel induces rapid bone regeneration in different clinical situations.

Neoligamentization process of BTPB used for ACL graft: histological evaluation from 6 months to 10 years.

Following anterior cruciate ligament (ACL) reconstruction with the middle third part patellar tendon, the graft undergoes histological rearrangement due to biomechanical action, which transforms it into a structure similar to the normal ACL. The purpose of our study was to make a qualitative and quantitative histological evaluation, by transmission electron microscopy (TEM), of the neoligamentization process of a bone-patellar tendon-bone (BTPB) graft used as pro-ACL at different follow-up times. We analysed the ultrastructure of collagen fibrils by focusing on their size and distribution with respect to a normal patellar tendon and a normal ACL used as controls. Our results showed that up to 24 months follow-up, progressive ultrastructural changes towards the normal ACL were observed. At longer times after surgery (48 and 120 months) no further changes were evident and the ultrastructure showed a marked reduction in large fibrils, which was typical of the control patellar tendon, and a significant increase in small fibrils. The ultrastructure seemed to combine fibrils from two different morphological units. The BPTB graft used as ACL underwent a transformation process for up to two years. After that period the transformation ceased and for ten years failed to reach the ultrastructural aspect of a normal ACL. However, from an architectural point of view the graft was slowly transformed into a structure similar to ACL with respect to the different mechanical stresses the ligament has to sustain.

Early detachment of titanium particles from various different surfaces of endosseous dental implants.

Titanium (Ti) endosseous dental screws with different surfaces (smooth titanium--STi, titanium plasma-sprayed-TPS, alumina oxide sandblasted and acid-etched--Al-SLA, zirconium oxide sandblasted and acid etched--Zr-SLA) were implanted in femura and tibiae of sheep to investigate the biological evolution of the peri-implant tissues and detachment of Ti debris from the implant surfaces in early healing. Implants were not loaded. Sections of the screws and the peri-implant tissues obtained by sawing and grinding were analysed by light microscopy immediately after implantation (time 0) and after 14 days. All samples showed new bone trabeculae and vascularised medullary spaces in those areas where gaps between the implants and host bone were visible. In contrast, no osteogenesis was induced in the areas where the implants were initially positioned in close contact with the host bone. Chips of the pre-existing bone inducing new peri-implant neo-osteogenesis were surrounded by new bone trabeculae. The threads of some screws appeared to be deformed where the host bone showed fractures. Ti granules of 3-60 microm were detectable only in the peri-implant tissues of TPS implants both immediately after surgery and after 14 days, thus suggesting that this phenomenon may be related to the friction of the TPS coating during surgical insertion.

Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients.

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

Histomorphometric, biochemical and ultrastructural changes in the aorta of salt-loaded stroke-prone spontaneously hypertensive rats fed a Japanese-style diet.

BACKGROUND AND AIM: It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone spontaneously hypertensive rats (SHRsp), concomitant salt loading and a Japanese-style diet greatly accelerate hypertension and the appearance of cerebrovascular lesions by directly damaging arterial vessels. A number of studies have characterised medium and small vessel lesions in SHRsp, but little attention has been paid to the changes in the wall structure of large arteries induced by exposure to a salt-enriched diet. The aim of this study was to investigate the effects of a Japanese-style diet and salt loading on the thoracic aorta. METHODS AND RESULTS: Two-month-old SHRsp were kept on a Japanese-style diet with 1% sodium chloride solution replacing tap water. Two months later, they were sacrificed and compared with age-matched or two-month-old control SHRsp kept on a standard diet and tap water in terms of the histomorphometry, ultrastructure and biochemical composition of the thoracic aorta. The vessel was consistently thicker in the four-month-old SHRsp (+20%, p < 0.05 vs two-month-old rats) regardless of diet. The salt-loaded SHRsp showed a significant reduction in elastic fibre density (-20%, p < 0.05 vs two-month-old rats) and an increase in the other matrix components (%), whereas the four-month-old controls showed preserved elastic fibres and a significant increase in the other matrix components (+65%, p < 0.05 vs two-month-old rats). There was a considerable increase in the amounts of 4-OH-proline (+147%), 5-OH-lysine (+174%) and desmosines (+360%) in the four-month-old controls vs their two-month-old counterparts (p < 0.01), but not in the salt-loaded animals. Ultrastructural analysis revealed clear damage and accelerated aging in the thoracic aorta of the salt-loaded SHRsp. CONCLUSIONS: Salt loading and a Japanese-style diet destabilize thoracic aorta architecture in SHRsp after two months of treatment.

Detachment of titanium and fluorohydroxyapatite particles in unloaded endosseous implants.

The shape, surface composition and morphology of orthopaedic and endosseous dental titanium implants are key factors to achieve post-surgical and long-term mechanical stability and enhance implant osteointegration. In this study a comparison was made between 12 titanium screws, plasma-spray-coated with titanium powders (TPS), and 12 screws with an additional coating of fluorohydroxyapatite (FHA-Ti). Screws were implanted in the femoral and tibial diaphyses of two mongrel sheep and removed with peri-implant tissues 12 weeks after surgery. The vibrational spectroscopic, ultrastructural and morphological analyses showed good osteointegration for both types of implants in host cortical bone. The portion of the FHA-Ti implants in contact with the medullary canal showed a wider area of newly formed peri-implant bone than that of the TPS implants. Morphological and EDAX analyses demonstrated the presence of small titanium debris in the bone medullary spaces near the TPS surface, presumably due to the friction between the host bone and the implant during insertion. Few traces of titanium were detected around FHA-Ti implants, even if smaller FHA debris were present. The present findings suggest that the FHA coating may act as a barrier against the detachment of titanium debris stored in the medullary spaces near the implant surface.

Familial atrophia maculosa varioliformis cutis: an ultrastructural study.

Atrophia maculosa varioliformis cutis is a rare and distinctive form of idiopathic facial macular noninflammatory atrophy that may rarely be observed in members of the same family. We describe two brothers, ages 14 and 16 years, with spontaneously appearing, asymptomatic, varioliform and linear atrophic lesions. Their past medical history was positive for varicella occurring in childhood without residual facial scarring. Routine laboratory investigations and screening for circulating autoantibodies were negative. Both patients were concordant for HLA A2 and DQ4.1. Routine and ultrastructural histologic examination of a punch biopsy specimen showed the presence of scarce, small, fragmented elastic fibers and compact collagen bundles associated with hypertrophic fibroblasts in the dermis. Our patients remained clinically stable, untreated, over a 2-year follow-up period. No long-term follow-up data have previously been reported.

Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia.

BACKGROUND: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta-thalassemia. OBJECTIVE AND METHODS: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. RESULTS: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta-thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta-thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. CONCLUSION: Data indicate that patients with beta-thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE).

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

Hyaluronan-phospholipid interactions.

Hyaluronan-phospholipid interactions have been studied in vitro by negative staining and rotary shadowing electron microscopy. Hyaluronan (HA) molecules of different molecular weights (around 170,000; 740,000, and 1.9 x 10(6) Da) were added to phospholipid suspensions (DPPC or egg lecithin) that were in the form of either unilamellar particles or multilamellar vesicles. Suspensions were then gently stirred and incubated at different temperatures from 24 hr up to 7 days. After 24 hr, at temperatures just above the melting point of the phospholipid used, both unilamellar particles and multilamellar vesicles were already shown to change their organization in the presence of HA, giving rise to the formation of (1) huge perforated membrane-like structures lying on the substrate; (2) 12-nm-thick "cylinders" (rollers) with a tendency to aggregate and to form sheets. These structures were seen only in the presence of high-molecular-weight HA, whereas low-molecular-weight HA (170 kDa) induced fragmentation of liposomes and formation of a few short rollers. These data show that phospholipids and HA interact and suggest they may also do so in vivo within the joint cavity, where both chemical species are present, giving rise to complexes which might exhibit peculiar lubricating and protective properties. It is also proposed that such interactions may not be as efficient in arthritic joints, where HA is degraded to low-molecular-weight fragments.

Ultrastructural identification of a membrane-like structure on the surface of normal articular cartilage.

Cytochemical and immunocytochemical approaches have been applied to the study of the surface of articular cartilage in humans, bovine and rats. Specimens were fixed in situ or soon after bioptic sampling with chemicals able to preserve and visualize proteins (glutaraldehyde, tannic acid), lipids (osmium tetroxide, malachite green, uranyl acetate) and proteoglycans (toluidine blue O, cuprolinic blue, cetyl pyridinium chloride). Mixtures of reagents were also used. Oriented serial thin sections were observed as such or after treatment with chemicals (chloroform-methanol, Triton X 100) or enzymes (chondroitinases, hyaluronidases, trypsin). Hyaluronan was detected by the use of glial-hyaluronate-binding-protein and antibodies against it. High concentration of osmium tetroxide or fixatives containing markers for lipid or for proteoglycans revealed that the surface of the articular cartilage, in all animal species examined, was covered by mono-multilayered discontinuous three-laminar sheets, which could be partly removed by chloroform-methanol and Triton X 100, were sensitive to hyaluronidase, chondroitinase and trypsin, and were immunopositive for hyaluronan. Each three-laminar sheet was 12-14 nm thick, was always separated from the cartilage itself and could be easily displaced. It is proposed that the surface of normal articular cartilage is covered by a discontinuous mono/multilayered pseudo-membrane, that can be better preserved by fixatives injected into the joint cavity and seems to consist of phospholipids, glycosaminoglycans and proteins. This membrane-like structure might have a protecting role in preventing direct contacts between the articular cartilage and toxic agents present in the synovial fluid and/or exert a lubricating effect within the articular joint.

Kinetic parameters of tyrosine hydroxylase in the rat's preoptic region during sleep deprivation and recovery induced by ambient temperature.

The role of catecholaminergic mechanisms in determining the changes in the rat's preoptic cyclic adenosine monophosphate (cAMP) concentration during sleep deprivation and recovery induced by ambient temperature was investigated in the present study. To this end, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was measured in the preoptic region of rats maintained in: (a) control (22 degrees C for 52 h), (b) deprivation (-10 degrees C for 52 h), and (c) recovery (22 degrees C for 4 h after 48 h at -10 degrees C) conditions. The enzyme followed a Michaelis-Menten kinetic. The analysis of substrate-related kinetic parameters (Km and Vmax) did not show any clear-cut difference between experimental conditions, which, as already known, induce both sleep deprivation and recovery in relation to significant cAMP changes.

The brain of the guinea pig in stereotaxic coordinates.

The brain of the guinea pig in stereotaxic coordinates is proposed as a tool for neurophysiologists who are interested in the guinea pig as an experimental animal. The frontal sections of guinea pig brain in stereotaxic coordinates extend from anterior (A) 17.0 to posterior (P) 5.0. The frontal sections of the brain are reproduced as drawings in which brain contours as well as nuclear and fibre structures are outlined. The procedure used for setting the stereotaxic coordinates and zero planes is reported and the limitation of the use of the tables is discussed.