Increased appearance rate of 27-hydroxycholesterol in vivo in hypercholesterolemia: a possible compensatory mechanism.

BACKGROUND AND AIMS: The first step in the alternative pathway of bile acid biosynthesis is the 27-hydroxylation of cholesterol, which takes place both in liver and extrahepatic tissues. This pathway is believed to play a role in peripheral cholesterol degradation. Aim of this study was to investigate the impact of hyperlipidemia on 27-hydroxycholesterol appearance rate, and to assess the effects induced by treatment with statins. METHODS AND RESULTS: Seven patients with familial hypercholesterolemia and eight patients with familial combined hyperlipidemia underwent determination of 27-hydroxylation rates in vivo by i.v. infusion of deuterated 27-hydroxycholesterol. Isotope enrichment was assayed by gas chromatography-mass spectrometry, allowing to calculate 27-hydroxycholesterol appearance rates. Six normocholesterolemic subjects were regarded as controls. In some hypercholesterolemic patients the infusions were repeated during treatment with atorvastatin or rosuvastatin. Hydroxylation rates were higher in hypercholesterolemic patients (8.7 ± 2.5 mg/h; controls, 3.4 ± 2.0 mg/h; combined hyperlipidemia, 4.4 ± 1.6 mg/h; mean ± SD, P < 0.01 vs both). After statin treatment, both plasma cholesterol levels and hydroxylation rates dropped by nearly 50%. No difference was detectable between the two statins. A linear correlation was shown between plasma cholesterol and 27-hydroxylation rates. CONCLUSION: Hypercholesterolemia associates with increased 27-hydroxycholesterol appearance rates, which decrease during hypocholesterolemic treatment. The correlation with cholesterol levels supports the view that 27-hydroxylation may act as a compensatory mechanism in a condition of larger plasma cholesterol pool. A regulatory role for hepatic and extrahepatic nuclear receptors seems reasonable. These data prompt novel pharmacological approaches for the management of hypercholesterolemia and the prevention of atherosclerosis.

In vitro cellular response and in vivo primary osteointegration of electrochemically modified titanium.

Anodic spark deposition (ASD) is an attractive technique for improving the implant-bone interface that can be applied to titanium and titanium alloys. This technique produces a surface with microporous morphology and an oxide layer enriched with calcium and phosphorus. The aim of the present study was to investigate the biological response in vitro using primary human osteoblasts as a cellular model and the osteogenic primary response in vivo within a short experimental time frame (2 and 4 weeks) in an animal model (rabbit). Responses were assessed by comparing the new electrochemical biomimetic treatments to an acid-etching treatment as control. The in vitro biological response was characterized by cell morphology, adhesion, proliferation activity and cell metabolic activity. A complete assessment of osteogenic activity in vivo was achieved by estimating static and dynamic histomorphometric parameters at several time points within the considered time frame. The in vitro study showed enhanced osteoblast adhesion and higher metabolic activity for the ASD-treated surfaces during the first days after seeding compared to the control titanium. For the ASD surfaces, the histomorphometry indicated a higher mineral apposition rate within 2 weeks and a more extended bone activation within the first week after surgery, leading to more extensive bone-implant contact after 2 weeks. In conclusion, the ASD surface treatments enhanced the biological response in vitro, promoting an early osteoblast adhesion, and the osteointegrative properties in vivo, accelerating the primary osteogenic response.

Nuclear receptors as potential molecular targets in cholesterol accumulation conditions: insights from evidence on hepatic cholesterol degradation and gallstone disease in humans.

The liver plays a central role in the regulation of cholesterol homeostasis. Hepatic cholesterol content is maintained by a complex interplay between input and output pathways; alterations in the balance among these processes may lead to accumulation of excess cholesterol in body compartments with potentially deleterious consequences at the level of blood vessels (atherosclerosis) and biliary tract (gallstone disease). Molecular biology has brought new insights into this field. Nuclear receptors have been shown to play a key role in the "sensing" of intracellular cholesterol levels and in the triggering of metabolic responses via the sterol regulatory element binding protein (SREBP) cascade. A nuclear receptor for bile acids, farnesoid X receptor (FXR), has been identified and the molecular pathways underlying feedback inhibition of bile acid synthesis, the main mechanism of irreversible degradation of cholesterol, have been clarified. Such regulation involves a number of additional coactivators/corepressors of the transcription of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase. Finally, the main transporters of biliary lipids (bile acids, phospholipids and cholesterol) have been described; most of them undergo transcriptional control by nuclear receptors, allowing regulation of biliary lipid efflux in conditions of different intracellular availability. Despite a body of evidence coming from experimental models the intimate mechanisms of regulation have not been clearly defined and direct evidence in humans is rather limited. This review will focus on the role of nuclear receptors in the regulation of hepatic cholesterol degradation and biliary lipid secretion, and on the theoretical applications from a pharmacotherapeutic perspective.

Age-related changes in bile acid synthesis and hepatic nuclear receptor expression.

BACKGROUND: Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. DESIGN: Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. RESULTS: Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. CONCLUSIONS: Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.

In vivo assessment of the osteointegrative potential of phosphatidylserine-based coatings.

The successful implantation of titanium-based implants for orthopaedic and dental applications is often hindered because of their mobility, which arises because of a lack of direct binding of the metal surface to the mineral phase of the surrounding bone. Ceramic coatings, although ensuring the integration of the implant within the tissue, are unstable and carry risks of delamination and of failure. Recently, a novel biomimetic approach has been developed where porous titanium implants are coated with calcium-binding phospholipids able to catalyse the nucleation of discrete apatite crystals after only 30 min incubation in simulated body fluids. The present work assesses the osteointegrative potential of this new class of coatings in an in vivo rabbit model and compares its performance with those of bare porous titanium and hydroxyapatite-coated titanium. The data obtained show that phosphatidylserine-based coatings, whilst resorbing, drive the growing bone into apposition with the metal surface. This is in contrast to the case of bare titanium.

Decreased hepatic expression of PPAR-gamma coactivator-1 in cholesterol cholelithiasis.

BACKGROUND: Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. MATERIALS AND METHODS: Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. RESULTS: No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). CONCLUSION: These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.

Review article: hyperlipidaemia and cardiovascular risk.

Hyperlipidaemia represents a determinant for the development of atherosclerosis and an important risk factor for cardiovascular disease, particularly in the context of the insulin resistance syndrome. This is characterized by alterations in the profile of plasma lipoprotein including high triglyceride levels, low high-density lipoprotein cholesterol concentrations and the appearance of qualitatively modified, small-dense low-density lipoproteins. Many charts and algorithms have been developed to estimate the entity of coronary and cardiovascular risk as related to dyslipidaemia, on the basis of additional individual risk factors and conditions: most include age and gender, smoking status, hypertension and diabetes. They should preferably be utilized in consistent patient populations, in terms of geographical areas and general risk profile. Pharmacological treatment of dyslipidaemia, in particular with statin drugs, was shown to greatly improve cardiovascular morbidity and mortality. A body of evidence also underlines the need for a multidisciplinary approach, integrating non-pharmacological lifestyle and diet interventions, as well as treatment of concomitant diseases (hypertension and diabetes).

Silybin, a new iron-chelating agent.

Silybin, a natural occurring flavolignan isolated from the fruits of Silibum marianum, has been reported to exert antioxidant and free radical scavenging abilities. It was suggested to act also as an iron chelator. The complexation and protonation equilibria of the ferric complex of this compound have been studied by potentiometric, spectrophotometric and electrochemical techniques. The formation of the complex silybin-Ga(III) in anhydrous DMSO-d6 has been studied by 1H NMR spectroscopy. Mass spectrometry and infrared spectroscopy on silybin-Fe(III) complex confirm all data obtained by 1H NMR spectroscopy. The experimental results show that silybin binds Fe(III) even at acidic pH. Different ternary complexes were observed at increasing methoxide ion concentration and their stability constants have been calculated. The results show the possible role of silybin in relation to the chelation therapy of chronic iron overload, as occurs in the treatment of Cooley's anemia.

Bone response to polymers based on poly-lactic acid and having different degradation times.

Authors studied two degradable and resorbable polymers derived from lactic acid: poly-L-Lactic acid (PLLA), with a relatively long time of degradation (longer than 6 months, PL10 Purac NL); poly-DL-Lactic acid (PDLLA), with a relatively short time of degradation (shorter than 6 months, PDL Purac NL). The animal species was the young adult New Zealand White rabbit. The in-vivo study was performed by implantation of small cylinders of 10 x 3 mm in size (length x diameter) in the distal metaepiphysis of the femur; 34 cylinders have been implanted. Retrievals of PLLA specimens took place at 3, 6, 9, 12 and 24 months; for PDLLA specimens at 1, 2, 4 months. Polarized light microscopy of undecalcified tissue sections was performed. The analysis for PLLA and PDLLA has shown a favorable response of bone tissue: alterations in the bone repair, growth and remodeling have not been observed. PLLA is persistent at the times studied; there is never a tight apposition between bone and PLLA implant and an intervening fibrous layer has often been observed. PDLLA is not persistent at the times studied and it degrades quite fast; bone repair of the empty implantation's hole occurs by bony growth from the endosteal trabeculae. The newly formed bone covers the hole's walls with an elongation parallel to them. For both polymers, whether the degradation is fast or slow, the material's substitution by newly formed bone never starts from the walls of the implantation hole. Only after the complete disappearance of the polymeric material newly formed bone begins to fill the hole.

Comparison in in-vivo response between a bioactive glass and a non-bioactive glass.

The authors report on the in-vivo comparison, in the rabbit, between the response to a bioactive glass and the response to a non-bioactive glass. Implants have been performed in muscle and bone. Two different glasses were investigated, namely B01 and I02. B01 is a glass designed to be degradable and resorbable and has a percentual molar composition of: SiO2 49.6%; P2O5 2.7%; CaO + MgO + Na2O + K2O + Al2O3 47.7% with a 1 : 1 CaO/Na2O ratio. I02 is a sodium-calcium-silicate non-resorbable glass lacking P2O5 and has a percentual molar composition of: SiO2 70.7%; CaO + MgO + Na2O + K2O + Al2O3 29.3%. In-vivo tests were planned as: (a) intramuscular implants of glass cylinders in the rectus femoris and retrievals took place at 2, 16 and 43 weeks; (b) intraosseus implants of glass cylinders in the distal femural canal and retrievals took place at 8 and 43 weeks. Histology and light microscopy analysis followed. Bioactive degradable glass elicits a favorable response both in muscle and bone; a gradual degradation process leads to disruption and partial resorption of the material and a tight apposition is promoted with the newly formed bone. The non-bioactive sodium-calcium-silicate glass (named I02) may elicit, like the bioactive degradable B01, a favorable response which is characterized by the absence of inflammatory or other adverse reactions; anyway it does not change its structure at an optical microscopic level and it does not promote any tight apposition with bone.

Response to polyetherimide based composite materials implanted in muscle and in bone.

The in-vivo response to a composite material obtained with polyetherimide (PEI) reinforced with carbon/glass fibers was investigated by histological methods by implanting cylinders in muscle and in bone of the New Zealand White rabbit. A common metallic alloy, widely used in orthopaedic surgery, was used as control (Stellite). The aim of the study was to analyze the biological response towards the surface of the material. Composite implants and metallic implants did not induce adverse or inflammatory reactions. The morphological picture produced was similar, in muscle and in bone, for both materials. In muscle, cylinders were confined by an extremely thin fibrous layer and the overall appearance of the muscular tissue was normal. In bone, cylinders were confined by a nearly annular rim of newly formed bone. From these data it is possible to derive that the response to PEI-based composite material is comparable with the response to metallic substrate and, then, the material can be suitable for clinical application.

X-ray diffraction and polarizing optical microscopy investigation of the structural organization of rabbit tibia.

X-ray diffraction and polarized optical microscopy investigations were carried out on thin sections of rabbit tibia in order to study the morphological organization of the structural components of this tissue, which often is utilized to test bone response to implants. In the optical microscope, the lateral face as well as the lateral portion of the caudal face exhibit a lamellar structure with an alternation of dark and bright lamellae running parallel to the long axis of the tibia. In contrast, both in the medial face and in the medial portion of the caudal face there are numerous osteonic structures. In spite of the complexity of this morphological organization, the results of small- and high-angle X-ray diffraction analyses indicate that the structural relationship between collagen fibrils and inorganic crystals is quite similar to that observed in single osteons and allows evaluation of the orientation of the two main structural components. Both collagen fibrils and apatitic crystallites are preferentially oriented parallel to the long axis of the tibia. The degree of orientation is greater in the thickness than in the plane of the lamellae, suggesting that collagen fibrils and inorganic crystallites lie preferentially in the plane of the lamellae, where they follow an oblique course. The degree of orientation of the apatitic crystallites is higher in the lateral face than in the medial and caudal faces, in agreement with the optical microscopic images. The results provide information that must be taken into account when evaluating the structural modifications of bone due to the insertion of a prosthetic device.

Bioactive glass coating: physicochemical aspects and biological findings.

Glass coating material was investigated before and after spraying to see whether it maintained the chemical and physical properties; in vivo and in vitro studies were done to evaluate the biological results. Following the spraying process, the Biovetro coating on the TiAl6V4 plate--as evidenced from chemical and physical analysis--maintains the properties of the original glass unchanged as far as the amorphous structure and its behaviour in a hydrolytic environment are concerned. In vitro and in vivo studies underline the positive features of the coating obtained by the plasma spray process, confirming that it has the typical properties of bioactive glass patented under the trade mark, Biovetro, i.e. biodegradability and osteoconductivity already confirmed by previous experimental protocols carried out by our group using powdered and fibre Biovetro.

Sodium fluoride sustained-release bone cement: an experimental study in vitro and in vivo.

A new "low temperature" bone cement, consisting of polymethylmethacrylate (PMMA) and 6% sodium fluoride, was developed for use in orthopedics. Fluoride is a well-known agent that may stimulate osteoblast activity and differentiation in vitro and in vivo; for this reason fluoride has been used for 30 years in the treatment of osteoporotic diseases of the bone. A local effect obtained with a slow release of fluoride from bone cement at the interface between bone and prosthetic implants could potentially enhance new bone formation around the prosthesis. This material was investigated both "in vitro", by establishing the kinetics of fluoride release from the acrylic bone cement and its maximal compressive strength, and "in vivo", by fitting 24 rabbits, which were then killed after 4, 12 and 16 weeks, with femoral implants, following labelling with fluorescent stains to allow the histologic evaluation of bone remodelling. The "in vitro" study revealed the release kinetics of fluoride from bone cement and the compressive strength of PMMA, that is not affected by the addition of fluoride. The "in vivo" investigation showed considerable healing ability after surgical and heat trauma, and new bone formation that appears larger on the surface in contact with fluoridated cement.

Thermal behaviour of hydroxyapatite intended for medical applications.

Four commercial hydroxyapatites (both natural and synthetic) were tested to assess transformations of the chemical and crystalline structure following variation of temperature from 20 to 1600 degrees C. The thermal behaviour of hydroxyapatite is relevant for biomedical applications such as plasma spraying of metallic implants. Thermogravimetric analysis showed a weight loss from each hydroxyapatite specimen, due to a release of structural H2O molecules; all the specimens up to 1300 degrees C were made of crystalline hydroxyapatite, determined by X-ray diffraction; at 1470 degrees C they were made of both hydroxyapatite and calcium phosphate, but at 1570 degrees C of calcium phosphate exclusively. The diffractograms of the hydroxyapatite coatings showed the same peaks as the original powders, so at the chosen plasma-spray procedure level no new phases were formed. The peak height was nevertheless lower in the plasma-sprayed hydroxyapatites for all interplanar spacing values, which indicated a lower degree of crystallinity, associated with a random structure derived from an alteration to the original crystalline network.

Comparative anatomical and ultrastructural features of the sensory papillae in the tongue of hibernating bats.

The papillae of the tongue dorsal surface of the insectivorous, hibernating bats (Vespertilionidae and Rhinolophidae), whose function is mainly sensorial, consist of two circumvallate papillae, two foliate papillae, located at the side edges at the glossopalatine arch, and numerous fungiform papillae. The circumvallate and foliate papillae are characterized not only by their position, but also by presence of several taste buds which open through the external orifice of the gustatory canal into the cavity of the vallum, or furrow, which divides the two folds of the lingual mucosa. The fungiform papillae (extremely numerous on the whole dorsal surface) are characterized by an unusual arrangement (along 3 oblique lines on the anterior two-thirds and predominantly on the middle line of the tongue body) and by the presence of only one to three taste buds which open on the heavily keratinized dorsal epithelial surface. The taste buds are made up of sensory cells with a light or dark matrix; their apical cytoplasmic expansions are not found beyond the middle part of the gustatory canal, in contrast with the circumvallate and foliate papillae which protrude from the orifice of the gustatory pore. Comparisons with the papillae of other types of bats and Insectivora and evaluations of the morphological characteristics and their functional values (unusual areas of distribution of the papillae, apical cytoplasmic expansions and behaviour of microfolds observed under SEM) have been made in different environmental conditions and nutritional habits, with attention to the mechanical events in the course of feeding.

Biocompatibility and osteogenetic characteristics of new biocompatible glasses.

A fibre-shaped glass with qualities of biocompatibility and biodegradation could be promising for reconstructive bone surgery in orthopaedics and neurosurgery. New highly biocompatible glasses, originally made by the Italian Group of Study on Biocompatible Glass, are obtained both in cylinder and fibre shapes due to their original chemical composition (Glassfiber). This is the only glass which is also available as a continuous filament. Hydrolytic in vitro and in vivo tests demonstrate that these glasses present both good biocompatibility and adjustable biodegradation in relation to their chemical composition. The advantages of bioactive glass also being available as a continuous filament are suggested.

Study of the osteoconductive properties of bioactive glass fibers.

Bioactive glass fibers have been prepared and implanted in cortical defect and in muscle. The fibers can act as a substrate for bone apposition, when implanted in a cortical defect, and become incorporated in the new bone matrix. The same results were obtained when fibers were implanted in a muscle pouch together with bone marrow cells. An intense inflammatory reaction was observed when bioactive glass fibers were implanted in muscle; the reaction was milder when fibers were implanted in bone or in muscle together with bone marrow cells. This fact supports the hypothesis that osteogenic cells adhere in an early phase to the substrate and prevent recognition of the foreign material by inflammatory cells. This appears to be a fundamental condition for direct bone matrix apposition on the surface of fibers.

Course and termination of the pyramidal tract in the pig.

To study the pyramidal tract in the pig, the motor cerebral cortex of one side was defined electrophysiologically and subsequently excised. The animals operated were killed after 7, 11 and 14 days, and the cerebral hemisphere of the operated side, brain stem and spinal cord were removed for histological examination. The pyramidal tract proved to run ipsilaterally as far as the oral extremity of the 12th cranial nerve nucleus. The decussation, which exhausted itself almost completely at the level of the rostral extremity of the 1st cervical metamere, started here. After the limit just mentioned only rare isolated fibres were visible. Along its course, the pyramidal tract sent a small number of axons to the ipsilateral and contralateral nucleus of the 7th cranial nerve, while the fibres running from the opposite side to the reticular formation and to the hypoglossal nerve nucleus, cuneatus, gracilis and trigeminal spinal tract nuclei were more numerous.