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PubMed2006

Subcutaneous evaluation of RF magnetron-sputtered calcium pyrophosphate and hydroxylapatite-coated Ti implants.

The in vivo behavior of infrared-heated, RF magnetron-sputtered hydroxylapatite (HA) and calcium pyrophosphate (DCPP) coated titanium discs was investigated. The discs were implanted subcutaneously in the back of six goats for 2, 4, 8 and 12 weeks. At the end of the study, coated discs were removed and examined on their physicochemical properties by X-ray diffraction (XRD) and scanning electron microscopy (SEM), including energy dispersive spectroscopy (EDS). Also, implants were prepared for light microscopical evaluation of the tissue response. The results showed that heat-treated HA coatings showed a stable behavior, i.e. no changes in the XRD pattern occurred during implantation. Also, no dissolution of the coating was observed by SEM. EDS revealed that the Ca/P ratio of the HA coatings remained stable during implantation. In contrast, heat-treated DCPP coatings showed a compositional change into apatite and tricalcium phosphate (TCP) during implantation. This was confirmed by the SEM and EDS analysis. The Ca/P ratio of the DCPP coatings changed from 0.8 to 1.52 during implantation. Finally, histology showed that both heat-treated HA and DCPP coatings showed no adverse tissue response, as characterized by the presence of thin, dense fibrous tissue capsule. Consequently, it can be concluded that 2 mum thick heat-treated, RF magnetron-sputtered HA and DCPP coatings are of sufficient thickness to withstand dissolution during 12 weeks of implantation in a subcutaneous location in goats. In addition, both coatings showed a biocompatible tissue behavior. Further, heat-treated DCPP coatings revealed a gradual compositional change into apatite and TCP.

Animals↗
PubMed2006

Macrophage-mediated biodegradation of poly(DL-lactide-co-glycolide) in vitro.

Biodegradation of poly-DL-lactide-co-glycolide (PLGA) both in vitro and in vivo has been well documented. However, the roles that macrophages and their fused multinucleated giant cells (MNGCs) play in this biodegradation are still unclear. The current study aimed to investigate macrophage-mediated biodegradation of PLGA thin films and of PLGA composites with hydroxyapatite (HA) and tricalcium phosphate (TCP) ceramic powders in vitro using a murine macrophage cell line (RAW 264.7). The interactions were analyzed by using cell viability assays, scanning electron microscopy, and focused ion beam microscopy. The results showed that RAW 264.7 cells effectively attached and proliferated on the PLGA films and PLGA-HA, PLGA-TCP composites. The RAW 264.7 cells were observed to aggregate and fuse to form MNGCs. The cell processes on the membrane, or pseudopodia, penetrated into the PLGA films and evidently eroded the surface. We conclude that macrophages and fused MNGCs actively respond to PLGA films as substratum and degrade the surface of this polymer.

Animals↗
PubMed2007

Peri-implant bone formation and implant integration strength of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer network-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly (acrylamide-co-ethylene glycol/acrylic acid) functionalized with an -Arg-Gly-Asp- (RGD) containing 15 amino acid peptides, derived from rat bone sialoprotein (bsp-RGD(15), were grafted to titanium implants in an effort to modulate bone formation in the peri-implant region in the rat femoral ablation model. Bone-implant contact (BIC) and bone formation within the medullary canal were determined using microcomputed tomography at 2 and 4 weeks postimplantation. BIC for bsp-RGD(15)-IPN implants was enhanced relative to hydroxyapatite tricalcium phosphate (HA-TCP) coated implants, but was similar to all other groups. Aggregate bone formation neither indicated a dose-dependent effect of bsp-RGD(15) nor a meaningful trend. Mechanical testing of implant fixation revealed that only the HA-TCP coated implants supported significant (>1 MPa) interfacial shear strength, despite exhibiting lower overall BIC, an indication that bone ingrowth into the rougher coating was the primary mode of implant fixation. While no evidence was found to support the hypothesis that bsp-RGD(15)-modified IPN coated implants significantly impacted bone-implant bonding, these results point to the lack of correlation between in vitro studies employing primary osteoblasts and in vivo wound healing in the peri-implant region.

Acrylates↗
PubMed2007

Vertical bone augmentation with granulated brushite cement set in glycolic acid.

Brushite cements are a biocompatible materials that are resorbed in vivo. A new cement composed of a mixture of monocalcium phosphate (MCP) and beta-tricalcium phosphate (beta-TCP) that sets using glycolic acid (GA) was synthesized and characterized. After setting, the cement composition, derived from X-ray diffraction, was 83 wt % brushite and 17 wt % beta-TCP with an average brushite crystal size of about 2.6 +/- 1.4 microm. The cement has a diametral tensile strength of 2.9 +/- 0.7 MPa. Granules prepared from the set-cement were used as grafting material in bone defects on rabbit calvaria for evaluating in vivo its bone regeneration capacity. Considerable cement resorption, improvement in the bone mineral density, and bone neoformation was observed after 4 weeks of the granules' implantation.

Absorbable Implants↗
PubMed2006

Surface chemistry and biological responses to synthetic octacalcium phosphate.

Octacalcium phosphate (OCP) has been suggested as a precursor of biological apatite in bone, dentin, and cementum because its existence explains the nonstoichiometry of apatite crystals in their compositions. Synthetic inorganic calcium phosphate compounds have been used clinically to fill bone defects, and sintered hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), bone substitute materials, are known to be osteoconductive, with beta-TCP also being bioresorbable. Nonsintered synthetic OCP has been shown to enhance bone regeneration accompanied by conversion into hydrolyzed apatitic products in situ and biodegradation. The surfaces of the OCP implant and the converted apatite seem to be continuously exposed to biological constituents, such as extracellular matrices, inorganic biominerals, and cellular components. This article reviews the surface reaction of OCP implants and the biological responses, such as experimentally stimulated bone formation on synthetic OCP, the mechanism of OCP hydrolysis into apatite, and the adsorption of biomolecules onto OCP and the converted apatite, of particular interest in reactive bone induction with synthetic OCP implants.

Adsorption↗
PubMed1991

A method for obtaining a pure population of t6/t6 mouse embryos prior to developmental arrest.

Developmental delay, as the result of ovariectomy, causes mouse blastocyst embryos obtained from +/t6 inter se matings to separate into two distinct populations when placed into outgrowth medium. One population remains as free floating embryos for a significantly longer period of time than the other population. Based upon their phenotypic expression following attachment and outgrowth, the former population was considered to be composed entirely of t6/t6 embryos and the latter, to be composed of +/+ and +/t6 embryos (Nadijcka et al., '81). In the present study, two-dimensional gel electrophoresis identified the embryos which were delayed in attachment as t6/t6 embryos since they synthesize only p63/6.9a, a product of the Tcp-1a locus which is unique to t-haplotypes. The early attaching embryos, assumed to be +/+ and +/t6, synthesize both p63/6.9a and b. The p63/6.9b protein is coded for by Tcp-1b on the wild-type homologous chromosome. Control +/+ blastocyst embryos synthesize only p63/6.9b. The data show that t6/t6 embryos can be identified prior to their lethal period and, thus, subjected to comparative studies to determine the cause of their lethality. Developmental delay is the first method established to enable one to unambiguously identify t6/t6 embryos prior to developmental arrest.

Animals↗
PubMed1991

Non-competitive antagonists of N-methyl-D-aspartate prevent spontaneous neuronal death in primary cultures of embryonic rat cortex.

Primary cultures of embryonic rat cerebral cortex were treated after 17 days in vitro for 10 min with a single dose of the non-competitive antagonists of N-methyl-D-aspartate (NMDA) receptor MK 801, TCP, and GK 11. They were then maintained in vitro for 31, 59, or 73 days, and then processed for the immunocytochemical detection of neuron-specific enolase (NSE). Immunoreactive cells were counted in treated and control cultures, and it was found that, except at 31 days, treated cultures contained far more NSE immunoreactive cells than controls. Moreover, this effect was dose-dependent, since with both TCP and GK 11 neuron survival was significantly higher with, respectively, 20 microM and 5 microM than with the lowest concentration of 2.5 microM. We tentatively conclude that spontaneous neuron death occurring in primary cultures in vitro is at least partly related to the NMDA-associated Ca++ channel, since the common property of the molecules we used is to block this channel. The relevance of this mechanism of cell death in vitro to neuronal death in vivo is discussed.

Animals↗
PubMed2007

Release of gentamicin from a tricalcium phosphate bone implant.

The impregnation and elution of gentamicin antibiotic from a commercially available porous beta-tricalcium phosphate (TCP) bone implant material (Vitoss, Orthovita, Inc.) was investigated in vitro. Sustained local antibiotic release is an attractive method for the prevention of infection following surgery. The purpose of this study was to evaluate the use of the naturally forming clot that occurs within a porous tissue scaffold when combined with autologous blood or bone marrow aspirate (BMA) as a method for achieving controlled drug delivery. The diffusion of antibiotic from porous TCP scaffolds was studied using water, clotted blood, or BMA as impregnating fluids. Incorporation of the drug into the porous scaffold using clotted blood or BMA as a binder produced slowed release relative to aqueous impregnated and dried samples. Modifications were made to the elution method to simulate restricted diffusion due to surrounding clotted blood, tissue, or bone that would occur in vivo. These modified methods simulated release in a surgical site and showed long release profiles, with significant amounts of antibiotic being released for up to 2 weeks. We concluded that adding gentamicin with autologous BMA is a promising method of controlling drug release.

Animals↗
PubMed1991

Redox derivatives of tranylcypromine: syntheses, properties, and monoamine oxidase inhibitor activity of some chemical delivery systems.

Several brain-targeting chemical delivery systems (CDS) based on a dihydropyridine----pyridinium salt type redox system were synthesized for the monoamine oxidase (MAO) inhibitor tranylcypromine (TCP). The dihydronicotinate moiety was chemically attached to the amino group of TCP by either an amide or substituted carbamate linkages. Physicochemical studies of the new derivatives, including chromatographic Rm determinations, were performed. Only the substituted carbamate-type derivatives manifested an increased lipophilicity relative to the parent compound. In vitro oxidation stability studies were also performed on selected derivatives using a ferricyanide-mediated method. Results of this assay showed that the dihydropyridine-type derivatives oxidized to the respective quaternary salt forms with stabilities which empirically correlated with other effective CDSs. Preliminary in vivo studies performed in rats indicated that some of the new derivatives exerted significant biological activity.

Animals↗
PubMed1997

Improved sensitivity to overlapping multiplet signals in in vivo proton spectroscopy using a multiecho volume selective (CPRESS) experiment.

A method for volume selective proton spectroscopy is presented based on a multiecho sequence with short refocusing interval tcp. It is demonstrated, that by appropriate choice of tcp on the order of 4-6 ms, signals from overlapping multiplets like the glutamine and glutamate (Glu/Gln) resonances in spectra of the human brain are considerably increased compared with a conventional PRESS volume selection scheme. Thus proton spectra from J-coupled multiplet signals can be acquired with TE on the order of 20-30 ms avoiding the baseline problems arising at shorter echo times due to broad resonances. This allows to selectively acquire spectra from substances with longer T2 without the confounding effects from J-coupling occurring in conventional volume selection techniques.

Alanine↗
PubMed1993

MK-801, phencyclidine (PCP), and PCP-like drugs increase burst firing in rat A10 dopamine neurons: comparison to competitive NMDA antagonists.

Extracellular single-unit recordings were used to assess the effects of PCP and PCP-like drugs (MK-801 and TCP) on the burst firing of ventral tegmental A10 dopamine neurons in the rat. The effects of these noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists were compared to the potent and competitive NMDA antagonists CGS 19755 and (+/-)CPP, and to BTCP, a PCP-derivative possessing little affinity for the PCP binding site within the ion channel gated by NMDA. PCP, MK-801, and TCP produced dose-dependent increases in the firing rate, which were accompanied by increases in the amount of burst activity, the number of action potentials within a burst, and the conversion of nonbursty cells to bursty. However, the coefficient of variation, a measure of the regularity of firing, was not significantly altered. These predominately excitatory effects contrast with the inhibition of firing, decrease in bursting, and regularization of pattern produced by BTCP. CGS 197555 and (+/-)CPP failed to alter any of the measured parameters. Thus, the increase in firing rate and amount of burst activity of dopamine neurons produced by PCP and PCP-like drugs, and the resultant hyperdopaminergia within the mesolimbic-mesocortical regions, could underlie the psychotomimetic properties of these compounds. Moreover, this effect would not appear to be related to a loss of activity at the NMDA recognition site, as evidenced by the lack of effect of the competitive NMDA antagonists.

Action Potentials↗
PubMed1994

RTI-4793-14, a new ligand with high affinity and selectivity for the (+)-MK801-insensitive [3H]1-]1-(2-thienyl)cyclohexyl]piperidine binding site (PCP site 2) of guinea pig brain.

[3H]TCP, an analog of the dissociative anesthetic phencyclidine (PCP), binds with high affinity to two sites in guinea pig brain membranes, one that is MK-801 sensitive and one that is not. The MK-801-sensitive site (PCP site 1) is associated with NMDA receptors, whereas the MK-801-insensitive site (PCP site 2) may be associated with biogenic amine transporters (BAT). Although several "BAT ligands" are known that bind selectively to PCP site 2 and not to PCP site 1 (such as indatraline), these compounds have low affinity for site 2 (Ki values > 1 microM). Here we demonstrate that the novel pyrrole RTI-4793-14 is a selective, high affinity ligand for PCP site 2. We determined the IC50 values of RTI-4793-14 and several reference compounds [PCP, (+)-MK801 and indatraline] for PCP site 1 (assayed with [3H](+)-MK801), PCP site 2 (assayed with [3H]TCP in the presence of 500 nM (+)-MK801) and a variety of BAT-related measures ([3H]CFT binding to the DA transporter, [3H]nisoxetine binding to the norepinephrine transporter, [3H]dopamine uptake, [3H]serotonin uptake). In addition, we determined the ability of RTI-4793-14 to block NMDA responses in cultured hippocampal neurons under voltage clamp. (+)-MK801 had high affinity for PCP site 1 (4.6 nM) and potently inhibited NMDA-induced responses, but was much less potent in the BAT-related measures (IC50 s > 10 microM). PCP had high affinity at PCP site 1 (IC50 = 92 nM) and PCP site 2 (IC50 = 117 nM), and was moderately potent in all BAT-related measures except [3H]nisoxetine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Effect of specific inhibitors on the anaerobic reductive dechlorination of 2,4,6-trichlorophenol by a stable methanogenic consortium.

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20 mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.

Journal Article↗
PubMed1995

Cloning of the cDNA for a brain glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein.

Polyclonal antibodies (Ab's) were raised against a 43-kDa component of a protein complex that has ligand recognition sites similar to those of brain N-methyl-D-aspartate (NMDA) receptors. The Ab's were used to immunopurify from brain synaptic membranes a 60-kDa glycine (Gly), glutamate (Glu) and thienylcyclohexylpiperidine (TCP)-binding protein and to screen a rat hippocampal cDNA expression library. A 1.85-kb clone, pGlyBP, coding for a protein of 470 amino acids (52.7 kDa) was identified. Northern blot analyses performed on poly(A+) RNA from brain revealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb. E. coli transformed with the pGyBP expressed a protein that was recognized by the anti-43 kDa Ab's and had recognition sites for Gly, Glu and TCP. The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X2-C-X16-20-C-X5-11), and 2 regions homologous to the NR1 receptor protein.

Amino Acid Sequence↗
PubMed1997

Mapping of G2/M-phase prevalences of chaperon-encoding transcripts by means of a sensitive differential hybridization approach.

The sensitivity of the differential hybridization approach is significantly increased by the application of size-selected probes. RNA from elutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase-specific cDNA libraries in the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side electrophoresis of equal amounts of PCR-amplified cDNA and elution of probes from parallel gel sections. Such size-selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase-specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein, HSC 70; heatshock cognate protein, HSC 73; eta subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin-containing TCP-1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.

Animals↗
PubMed2001

Review: cellular substrates of the eukaryotic chaperonin TRiC/CCT.

The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containing TCP-1) is a large (approximately 900 kDa) multisubunit complex that mediates protein folding in the eukaryotic cytosol. The physiological substrate spectrum of TRiC is still poorly defined. Genetic and biochemical data show that it is required for the folding of the cytoskeletal proteins actin and tubulin. Recent years have witnessed a steady stream of reports that describe other proteins that require TRiC for proper folding. Furthermore, analysis of the transit of newly synthesized proteins through TRiC in intact cells suggests that the chaperonin contributes to the folding of a distinct subset of cellular proteins. Here we review the current understanding of a role for TRiC in the folding of newly synthesized polypeptides, with a focus on some of the individual proteins that require TRiC.

Animals↗
PubMed1996

Characteristics of vascular wall cells subjected to dynamic cyclic strain and fluid shear conditions in vitro.

We recently developed an in vitro silicone rubber tubular apparatus, the vascular simulating device (VSD), which simulates pressure, flow, and strain characteristics of peripheral arteries (Benbrahim et al., 1994, J. Vasc. Surg. 20, 184-194). In this report, we tested the ability of silicone rubber surfaces to support the growth and differentiation of endothelial cells (EC) and smooth muscle cells (SMC) and studied the effects of arterial levels of pressure, flow, and strain on these properties. Human umbilical and saphenous vein EC and bovine aortic EC and SMC were cultured on coated and uncoated silicone rubber in flat and tubular configurations (6 mm inner diameter) and on tissue culture plastic (TCP). Attachment, growth, and differentiation were compared on these surfaces. In addition, the effects of arterial pressure, flow, and strain conditions on adhesion and subsequent growth and differentiation were studied in the tubular configuration. Attachment and growth of vascular wall cells on fibronectin-coated silicone rubber was similar to that obtained on TCP. Application of arterial levels of pressure, flow, and strain did not alter adhesion of the cells to the tubes. Subsequent passage of these cells demonstrated that attachment, growth, and differentiation (uptake of LDL and expression of factor VIII-related antigen by EC and expression of muscle-specific actin by SMC) were similar in cells derived from experimental and control tubes which were not subjected to arterial conditions. Finally, mRNA expression of specific "housekeeping" genes was similar in cells isolated from experimental and control tubes. We conclude that the VSD supports the culture of viable and differentiated EC and SMC. These experiments demonstrate that it is possible to evaluate the effects of arterial strain and fluid shear on vascular wall cells in vitro, in a configuration similar to the blood vessel wall.

Aorta↗
PubMed1995

Thymine auxotrophy as an attenuating marker in Vibrio cholerae.

Vibrio cholerae CVD102 is a thymine-dependent auxotroph of CVD101, a cholera toxin A-B+ candidate live oral cholera vaccine. Previous clinical experience with these strains suggested that, by restricting intestinal growth, thymine auxotrophy is attenuating for V. cholerae. Studies in the infant mouse cholera model cast doubt upon this conclusion however. Stable thyA mutants selected from each of three pathogenic strains showed unimpaired gut colonization in mixed-infection competition experiments. Similar results were obtained using thyA mutants selected from two atoxigenic strains, including CVD101. Further studies with CVD102 showed that the reduced colonization potential of this strain could not be compensated by the provision of a functional thyA+ gene in trans. CVD102 shows reduced synthesis of toxin-coregulated pili (TCP) during in vitro growth, suggesting the presence of a second, undefined mutation in this strain. Given the critical role of TCP in intestinal colonization, it seems probable that this previously unrecognized mutation is responsible for the poor in vivo performance of CVD102.

Animals↗