The slow release of BMP-7 at a low dose accelerates dental implant healing in an osteopenic environment.

The aim of the present study was to investigate in vivo whether bone morphogenetic protein-7 (BMP-7) was able to promote and accelerate dental implant healing at a low dose in an osteopenic environment by using a delayed drug-release system. Skeletally mature Chinese goats, having physiologically osteopenic (osteoporotic-like) facial bones, served as an animal model. Dental implants were provided with a delayed-release drug-delivery system and BMP-7 was applied at three different dosages. The implants, inserted into healed extraction sockets, were removed 1, 2 and 3 weeks after surgery. Quantification of osseointegration and formation of new bone in the peri- implant space were measured histomorphometrically. Data revealed no evidence of any adverse drug effect at or near the implantation sites. After the first postoperative week, bone neoformation was minimal; after the second week, peri-implant bone formation appeared, particularly in the groups with low dosages of BMP-7. After 3 weeks, new-bone volume was the largest in the group with the lowest (near-physiological) dosage of BMP-7, also showing the highest efficacy of BMP-7. Other dosage or release modes were found to be significantly less effective. BMP-7 was highly efficacious in promoting and accelerating bone formation in the peri-implant space in a hostile osteopenic environment if released by a slow-mode mechanism over time at near physiological activities. Therefore, biological functionalisation of dental implants by a high-power osteogenic factor may improve their healing success in hostile bony environments (osteopenia, osteoporosis, bone atrophy etc.).

Optimisation of BMP-2 dosage for the osseointegration of porous titanium implants in an ovine model.

In clinical orthopaedics, total joint replacements and spinal fusions are routine undertakings. Many of the implicated patients suffer from osteoporosis, severe arthrosis or osteopaenia. In individuals thus afflicted, the bony bed lacks the mechanical stability that is a requisite for a firm anchorage of the implant and its functional competence. To promote the bony bondage of an implant it is necessary to induce neo-ossification by the introduction of an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2). Since this growth factor is generally applied in a free form and at high dosages to maximise its osteogenicity, untoward side effects frequently ensue. We hypothesise that the administration of BMP-2 using a suitable delivery vehicle, and its gradual, low dose release therefrom in a cell-mediated manner, would avert the triggering of undesired side effects and enhance its efficacy. To test this postulate, implants of porous titanium were coated with a layer of calcium phosphate into which BMP-2 was biomimetically incorporated at dosages ranging from 0.8 to 500 µg/g of coating material (delivery system) prior to their surgical placement in the tibiae of adult sheep. The volume and the surface area of newly-formed bone were evaluated histomorphometrically after 3 and 6 weeks. The highest values were achieved using BMP-2 dosages of 20 to 100 µg/g of coating: The deposition of bone was confined to the immediate vicinity of the implant and was observed deep within the interstices of its meshwork, to the walls of which it bonded well. The findings of the study attest to the validity of our hypothesis.

An educational review of cartilage repair: precepts & practice--myths & misconceptions--progress & prospects.

OBJECTIVE: The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN: Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS: Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-associated adaptations to biomechanical and anatomical roles.

OBJECTIVE: Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focusing on structural issues, which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. METHODS: Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20-40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy (EM) and unbiased stereological methods. Morphological variations disclosing an association between joint type (knee vs ankle) and biomechanical role (convex vs concave articular surfaces) were identified by a 2-way analysis of variance (ANOVA) and a post-hoc analysis. RESULTS: Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex vs concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2 × 10(6) chondrons/cm(3). CONCLUSION: Variation in cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help to identify conditions where OA is likely to occur.

Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor.

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-ß)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-ß1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-ß1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-ß1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-ß1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC(1)) receptor.

To study desensitization and glycosylation of the type I pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC(1)R), a hemagglutinin (HA) epitope was inserted within the N-terminal extracellular domain, allowing immunological detection of PAC(1)R both in intact and permeabilized cells. PAC(1)R was tagged without loss of functions in ligand binding and ligand-stimulated cAMP production. In transiently transfected COS-7 cells, PAC(1)R was localized both in the plasma membrane and the cytoplasm around the nucleus. By immunoblot analysis, the immunoreactive bands with relative molecular masses ranging from 45 to 70 kDa were detected in the membrane fractions of PAC(1)R-expressing COS-7 cells. Digestion of the membranes with endoglycosidase F or treatment of the cells with tunicamycin decreased the size of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC(1)R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearance of cell surface receptor. In accordance with this observation, preexposure of cells to PACAP38 induced a desensitization of PAC(1)R to the agonist response, although it did not cause a reduction in PAC(1)R mRNA or protein level and even slightly elevated them. These results suggest that agonist-induced desensitization of PAC(1)R involves the receptor sequestration.

Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.

Metalloproteinase inhibitor prevents hepatic injury in endotoxemic mice.

This study was conducted to examine of [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)-succinyl]-L- phenylalanine-N-methylamide (GI 129471), a matrix metalloproteinase inhibitor, for its effects on increase of serum pro-inflammatory cytokine levels as well as hepatic injury in D-galactosamine plus lipopolysaccharide-injected mice. In vitro experiments showed that GI 129471 was able to inhibit the elevation of tumor necrosis factor-alpha (TNF-alpha) in LPS-stimulated human and mouse whole blood with IC50 values of 370 nM and 260 nM, respectively. When administrated i.p. at 40 mg/kg, GI 129471 significantly reduced serum TNF-alpha level but not other pro-inflammatory cytokines in D-galactosamine plus lipopolysaccharide-injected mice. Treatment of mice with GI 129471 also reduced biochemical indices of hepatic injury to the normal level. Histopathological findings indicated that GI 129471 treatment can prevent severe centrilobular necrosis in liver. These results suggest that release of TNF-alpha from lipopolysaccharide-stimulated cells is the critical step leading to hepatic injury in endotoxemia and that a matrix metalloproteinase inhibitor with an inhibitory action on this step may be a promising drug for the clinical treatment of endotoxemia accompanied by hepatic injury.

Selective expression of a glutathione S-transferase subclass during spermatogenesis.

Levels of the hGSTM3 glutathione S-transferase (GST) subunit in testis of the human fetus and infant were found to be only a small fraction of those in adults. To understand these observations and to determine whether hGSTM3 subunit expression is developmentally and/or hormonally regulated, an experimental model based on the rat testis homologue (subunit rGSTM5) was used. For prepubertal rats, testicular rGSTM5 subunit levels were very low, but a sharp increase was observed between weeks 6 and 7 of development, when testicular growth includes increased numbers of germ cells associated with spermatogenesis. In adult hypophysectomized rats, the rGSTM5 subunit content of testis decreased progressively over 5 weeks, at which time the subunit was barely detectable. In contrast, the other GST subunit types did not vary significantly during development or after hypophysectomy. These results suggest that rGSTM5 subunits in rat testis could originate from spermatogenic cells. Accordingly, GSTs were purified from human sperm, and it was shown that the hGSTM3 subunit was, by a large measure, the predominant form. These data are consistent with the notion that the differential expression of hGSTM3 during human testicular development can also be explained on the basis of its preferential location in germs cells.

Human testicular glutathione S-transferases: insights into tissue-specific expression of the diverse subunit classes.

Cytosolic glutathione S-transferase (GST) subunits from human testis were resolved by HPLC and unambiguously identified by combined use of peptide sequence-specific antisera and electrospray ionization mass spectrometry (ESI MS). Allelic variants of hGSTP1, hGSTM1 and hGSTA2 were distinguished on the basis of observed differences in their molecular masses. Relative amounts of the multiple different subunit types in various human tissues were determined from HPLC profiles. From this type of analysis, tissues from hGSTM1 null allele individuals were readily discerned at the protein level; liver was the only tissue in which the hGSTM1 subunit was the major mu-class GST. hGSTM4 and hGSTM5 subunits were found at very low levels in all tissues examined. By far the tissue richest in the unique hGSTM3 subunit was testis, although brain also has significant levels.

[Fluctuations in serum antigens and antibodies in a patient with primary cutaneous aspergillosis associated with acute leukemia].

A 5-year-old girl developed cutaneous aspergillosis due to Aspergillus flavus while undergoing remission induction therapy for acute lymphocytic leukemia. Of six serum samples obtained during the acute stage of Aspergillus infection, four showed antigenemia (6.5-22.9 ng/ml) determined by enzyme-linked immunosorbent assay (ELISA). However, five serum samples obtained after treatment with amphotericin B and granulocyte-colony stimulating factor showed negative results for antigens. Sera obtained on day 17 after the detection of skin lesions showed seroconversion in precipitin antibody determined by an immunodiffusion test and in immunoglobulin (Ig) A class antibody determined by ELISA, while sera obtained on day 24 showed seroconversion of IgG and IgM class antibodies. The patient achieved complete remission of leukemia and was discharged on the 92nd day of hospitalization. No signs of disseminated or deep-seated fungal infections were present during the hospitalization. Assays for serum antigens may be of value for the early diagnosis of invasive aspergillosis. Moreover, persistently negative results for antigens in accordance with antibody responses may correlate with recovery from the infection.

[Clinical study of fluconazole-injectable and -granules in pediatric patients].

In this study, we have investigated the clinical effectiveness of fluconazole (FLCZ) given intravenously or orally to pediatric patients with systemic fungal infections. FLCZ was administered intravenously to two patients with acute leukemia (multiple hepatosplenic candidiasis and aspergillosis) and orally to two mycosis complicated with neuroblastoma and aplastic anemia, respectively. Clinical efficacies were excellent and no side effects were observed in any patients. Pharmacokinetic analysis in 6 neonates revealed that the plasma half-life is 37-41 hours after administration of single dose of intravenous infusion of 3 mg/kg of FLCZ.

[Expression and extracellular release of transferrin receptors on erythropoiesis].

One of the most important factors for the proliferation and hemoglobin synthesis of erythroid cells is iron atom. This atom is tightly bound to serum transferrin (Tf) and is taken up by erythroblasts and reticulocytes through transferrin receptor (TfR). Both Tf and TfR are reutilizable and have roles for the efficient intracellular accumulation of iron. In addition to the reutilization (recycling), the expression of TfR is also regulated by cytoplasmic iron concentration; the increase of iron downregulate the synthesis of TfR at the translational level and vice versa. This mechanism was recently explained by the binding between "iron responsive element (IRE)" in the 5' end of TfR mRNA and IRE binding protein by a transacting manner. Johnstone et al, and we found that TfR was externalized from sheep reticulocyte and human erythroleukemia cell, K562, respectively. Furthermore, we confirmed that this shed TfR was detected in blood and concluded that the quantitation of TfR in serum is a useful index for evaluating the erythropoiesis. The serum TfR was increased in iron deficiency anemia, hemolytic anemia and polycythemia and was decreased in aplastic anemia. In renal anemia, it was increased after the administration of erythropoietin (Epo). By the in vitro liquid culture of peripheral blood stem cells using interleukin 3 and Epo, it was found that soluble TfR was derived from the erythroblasts during the maturation process.

[Immunological effect of immunoglobulin on experimental colitis induced by dextran sulfate].

To clarify the mechanism of action of immunoglobulin (IgG) in intravenous immunoglobulin therapy for ulcerative colitis (UC) patients, we studied the effect of IgG on the dynamics of immunocompetent cells in the colonic mucosa of experimental colitis induced by dextran sulfate sodium (DSS) in rats. The administration of the same species' IgG suppressed the mucosal infiltration of immunocompetent cells (activated T cells, macrophages and neutrophils), although the different species' IgG didn't. We have already shown that the same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha, IL-1 alpha and IL-8) in the colonic mucosa of experimental colitis induced by DSS. In the present report, we demonstrated the different species' IgG, as well as same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha and IL-1 alpha) from lamina propria mononuclear cells of rat large intestine in vitro. Therefore, it was considered that the suppression of cytokine production was a consequence of the decreased immunocompetent cells in colitis mucosa. Furthermore, we demonstrated that the DSS-treated antigen presenting cells (APCs) activated antigen-specific T cells as a possible mechanism underlying the colitis induced by DSS and the same species' IgG inhibited this activation of T cells.

[Effect of gamma-immunoglobulin in experimental colitis induced by dextran sulfate].

To clarify the mechanisms of action of immunoglobulin (IgG) in intravenous immunoglobulin therapy for the ulcerative colitis (UC) patients, we studied the therapeutic effect of IgG on experimental rat colitis induced by dextran sulfate sodium and on the production of inflammatory cytokines such as TNF alpha, IL-1 alpha and IL-8. The administration of rat IgG demonstrated to suppress the development of blood stool and the induction of the ulcerative lesion in large intestine. The levels of TNF alpha, IL-1 alpha and IL-8 were increased in blood and mucosa of the large intestine in this model. Rat IgG showed a tendency to decrease the levels of the cytokines in colonic mucosa. This result seems to provide a piece of explanation of how massive administration of IgG shows an improvement in the ulcerative lesion of UC patients.

Central PACAP mediates the sympathetic effects of leptin in a tissue-specific manner.

We previously demonstrated that the peptidergic neurotransmitter pituitary adenylate cyclase-activating polypeptide (PACAP) affects the autonomic system and contributes to the control of metabolic and cardiovascular functions. Previous studies have demonstrated the importance of centrally-mediated sympathetic effects of leptin for obesity-related hypertension. Here we tested whether PACAP signaling in the brain is implicated in leptin-induced sympathetic excitation and appetite suppression. In anesthetized mice, intracerebroventricular (ICV) pre-treatment with PACAP6-38, an antagonist of the PACAP receptors (PAC1-R and VPAC2), inhibited the increase in white adipose tissue sympathetic nerve activity (WAT-SNA) produced by ICV leptin (2µg). In contrast, leptin-induced stimulation of renal sympathetic nerve activity (RSNA) was not affected by ICV pre-treatment with PACAP6-38. Moreover, in PACAP-deficient (Adcyap1-/-) mice, ICV leptin-induced WAT-SNA increase was impaired, whereas RSNA response was preserved. The reductions in food intake and body weight evoked by ICV leptin were attenuated in Adcyap1-/- mice. Our data suggest that hypothalamic PACAP signaling plays a key role in the control by leptin of feeding behavior and lipocatabolic sympathetic outflow, but spares the renal sympathetic traffic.

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) are more susceptible to retinal ischemic injury in vivo.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuroprotective peptide exerting protective effects in neuronal injuries. We have provided evidence that PACAP is neuroprotective in several models of retinal degeneration in vivo. Our previous studies showed that PACAP treatment ameliorated the damaging effects of chronic hypoperfusion modeled by permanent bilateral carotid artery occlusion. We have also demonstrated in earlier studies that treatment with PACAP antagonists further aggravates retinal lesions. It has been shown that PACAP deficient mice have larger infarct size in cerebral ischemia. The aim of this study was to compare the degree of retinal damage in wild type and PACAP deficient mice in ischemic retinal insult. Mice underwent 10 min of bilateral carotid artery occlusion followed by 2-week reperfusion period. Retinas were then processed for histological analysis. It was found that PACAP deficient mice had significantly greater retinal damage, as shown by the thickness of the whole retina, the morphometric analysis of the individual retinal layers, and the cell numbers in the inner nuclear and ganglion cell layers. Exogenous PACAP administration could partially protect against retinal degeneration in PACAP deficient mice. These results clearly show that endogenous PACAP reacts as a stress-response peptide that is necessary for endogenous protection against different retinal insults.

Comparative examination of inner ear in wild type and pituitary adenylate cyclase activating polypeptide (PACAP)-deficient mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with well-known neuroprotective and neurotrophic effects. The involvement of PACAP in sensory processing has also been documented, but little is known about its effects in the auditory system. PACAP and its specific receptor (PAC1) are present in the cochlea and in brain structures involved in auditory pathways. Recently, we have shown that PACAP protects cochlear cells against oxidative stress-induced apoptosis. The endolymphatic Ca(2+) concentration controlled by Ca(2+) buffers of the hair cells is essential for the normal hearing processes. In this study we examined the localization of PAC1 receptor and Ca(2+) buffering proteins (parvalbumin, calretinin, calbindin) in the inner ear of 5-day-old PACAP-deficient mice compared with wild-type mice in order to get a closer insight into the effect of endogenous PACAP in the cochlear function. We did not find differences in the distribution pattern of PAC1 receptors between the two groups, but wild-type animals showed significantly higher PAC1 receptor expression. In contrast, inner and outer hair cells of PACAP-deficient mice showed more pronounced parvalbumin, calbindin, and calretinin immunopositivity compared with wild-type mice. Elevated endolymphatic Ca(2+) is deleterious for cochlear function, while the high concentration of Ca(2+) buffers in hair cells may offer protection. The increased immunoreactivity of Ca(2+) binding proteins in the absence of PACAP provide further evidence the important role of PACAP in the hearing processes.

Role of endogenous pituitary adenylate cyclase-activating polypeptide in adult hippocampal neurogenesis.

Hippocampal neurogenesis occurs throughout life in mammals and has pivotal roles in brain functions. An enriched environment stimulates hippocampal neurogenesis, but the exact mechanisms are still unclear. The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult hippocampal neurogenesis under standard or enriched rearing conditions. Rearing in the enriched conditions from 4-weeks old for 4-weeks increased the survival of newly divided cells in the subgranular zone and granule cell layer of the dentate gyrus of wild-type and PACAP-knockout (PACAP-/-) mice. The increase in the survival in the granule cell layer was less in PACAP-/- mice than in the wild-type mice. In contrast, the proliferation of newly divided cells in mice reared in the standard and enriched conditions did not differ between the wild-type and PACAP-/- mice. Regarding the differentiation of newborn cells in the dentate gyrus, most of the newly divided cells exhibited the neuronal phenotype in both the wild-type and PACAP-/- mice under standard and enriched conditions. These findings suggest that endogenous PACAP is partly involved in the survival of the enriched environment-induced generation, but not in the basal rate, of newborn cells in the dentate gyrus of the adult hippocampus.