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An ultrastructural study of the process of hard tissue formation in amputated dental pulp dressed with alpha-tricalcium phosphate.

This study was undertaken to determine the reaction of amputated dental pulp to the capping agent, alpha-tricalcium phosphate (alpha TCP), and to reveal the exact nature of the processes involved in both the healing of, and new hard tissue formation in the pulp when dressed with alpha TCP. The dental pulps of the anterior teeth of Macaca fuscata were dressed with alpha TCP ceramic 4-10 microns in diameter. The teeth were fixed by perfusion, then observed with light and electron microscopes. At 10 and 14 days postoperatively, the phagocytosis of the alpha TCP layer by the many macrophages and multinucleated giant cells, both of which had emerged beneath it, was seen. The synthesis of collagenous matrix underlying the alpha TCP layer began at 14 or 21 days, the cells involved exhibiting morphologic characteristics similar to those seen in osteoblasts. By the 90th day, only a bone-like hard tissue remained. No inflammation was observed in the residual dental pulp at any time during the experimental period. At 14 days, needle-like crystals were seen to have formed on the surface of alpha TCP particles, and by the 21st day, these crystals had extended to the newly synthesized matrix, with the result that calcification of the matrix was initiated, a calcified layer being superficially produced in the matrix. In the region of the matrix adjacent to the superficial calcified layer, a number of matrix vesicles were also found to function as another mechanism of matrix mineralization. Thus, it was concluded that the bone-like hard tissue was induced in the pulp capped with alpha TCP, and its potential for clinical application to exposed dental pulp was confirmed.

Animals↗

[Purification and identification of pili of Vibrio cholera O139].

To provide the base of manufacturing V. cholera O139 subunit vaccine, purification and identification methods of V. cholera O139 Toxin-coregulated pili (TCP) were studied. The results showed the optimal expressing condition of TCP was in AKI or CFA medium, at 30 C for 24-36 h with stationary cultivation. The molecular weight of the TCP subunit protein measured by sodium dodecyl sulfate-poly acrylamide gel electrophoresis was about 20.5 kd. By using mouse anti-TCP monoclone antibody and sheep anti-mouse IgG-HRP antibody, the TCP was confirmed by dot blot immunoassay. Rabbit immune serum of TCP purified from O139 strain agglutinated both O139 biotype and El Tor biotype Vibrio cholera strains. These indicate that TCP may be an effective and protective antigen to conduct the preparation of V. cholera vaccine.

Animals↗

[A histologic observation of bone formation induced by subperiosteal implantation of resorbable porous block beta-tricalcium phosphate on calvarial surface in rabbits].

OBJECTIVE: By the histologic observation of samples, understanding the osteoconductive histologic behaviour and resorbable mechanism of beta-tricalcium phosphate (beta-TCP). METHODS: Porous block beta-TCP samples were implanted into calvarial surface. Tissue slices dyed with methods of HE and Masson's were observed by microscope and compared with hydroxyapatite(HA). RESULTS: Beta-TCP had the osteoconductivity,as well as HA, plentiful new bone penetrated into materials from the calvarial surface. There was no new bone under the periost obviously. A large number of rhagiorine cells swallowing beta-TCP had been observed. CONCLUSION: Beta-TCP has osteoconductivity, can be used as a substitution for atrophic alveolar ridge augmentation. The function of rhagiorine cell swallowing beta-TCP is the essential resorbable mechanism of beta-TCP.

English Abstract↗

Blood pressure effects of tranylcypromine when prescribed singly and in combination with amitriptyline.

Data on blood pressure was extracted from the findings of a 6-week double-blind study that was carried out to evaluate the efficacy and safety of prescribing tranylcypromine (TCP) singly and in combination with amitriptyline (AMI). The effect of TCP on blood pressure was one of the methods used to evaluate its safety. Target daily doses of medication for the final 2 weeks of the study were: TCP 30 mg, AMI 150 mg, or the two in combination. Because of side effects, mean daily doses actually achieved were lower: single TCP 18.5 mg; single AMI 114.3 mg; combined TCP/AMI: TCP 19.7 mg, AMI 108.7 mg. When TCP alone was prescribed, a significant fall in diastolic blood pressure in the standing position was noted and orthostatic hypotension was more frequently observed. Dizziness was complained of on at least one occasion in over three quarters of patients on TCP/AMI. No correlation between blood pressure effects and dizziness was detected and there were no other troublesome side effects from this antidepressant combination. The study concluded that although TCP's overall effect on mean blood pressure readings when prescribed alone or in combination was small, its orthostatic effect is noteworthy and should be borne in mind by the prescribing clinician.

Adult↗

Clinico-pathological studies on the tissue reactions of human pulp treated with various kinds of calcium phosphate ceramics.

The aim of this study was to evaluate the clinical values of calcium phosphate ceramics from the biological point of view. Two kinds of calcium phosphate ceramics, tricalcium phosphate (TCP) and hydroxyapatite (HAP) ceramics with a low sintering temperature (LT) and a high sintering temperature (HT), were applied to exposed pulp wounds in ninety healthy human teeth. Each group was further subdivided into 2 groups with selected granule diameters of 5 microns (S) and 40 microns (L), and investigated clinico-pathologically. Clinically, cases showing some kinds of discomfort symptoms comprised 3/15 of the TCP (S) group, 3/15 of the TCP(L) group, 3/15 of the LT(S) group, 4/15 of the LT(L) group, 2/15 of the HT(S) group, and 3/15 of the HT(L) group. The symptoms were generally slight or very slight and disappeared within eight days in the LT(S) group. Histo-pathologically, inflammatory reactions were observed within 2 weeks. Traumatic injury caused by surgical wound or by mechanical irritation from the materials produced various pathological changes. After the treatment, pulp manifested reparative changes such as cicatrization, formation of new denticles, and dentin apposition on pulp-chamber walls. In many cases, TCP and HAP produced similar effects on human dental pulp. In the TCP(S), the LT(S), and HT(S) groups, a calcified matrix had appended by 3 weeks. In TCP(L), LT(L), and HT(L) groups, the matrix had appended from 3 to 6 weeks. Calcified matrix formation in the LT group was slightly less than in that in the HT of HAP group. In a few cases, complete dentin bridges formed over the exposed pulp surfaces. These results suggest that HAP (both LT and HT) and TCP ceramics are biocompatible and do not harm human dental pulp. These ceramics were found to be clinicopathologically effective in biologically protecting exposed human dental pulp and useful as basic materials in endodontic therapy.

Adolescent↗

Evidence that zinc inhibits N-methyl-D-aspartate receptor-gated ion channel activation by noncompetitive antagonism of glycine binding.

Zinc noncompetitively antagonizes N-methyl-D-aspartate (NMDA) receptor-mediated responses in cultured neurons. We investigated the mechanism of this inhibition by examining the effect of zinc on ligand binding to three distinct sites on the NMDA receptor in rat hippocampal membranes. Zinc dose-dependently inhibited both the association and dissociation of the NMDA channel blocker [3H]N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) but had no effect on steady state levels of [3H]TCP binding. This suggests that zinc inhibits the receptor-gated access of [3H]TCP to its site in the ion channel but has no effect on the binding site itself. Zinc inhibition of [3H]TCP association was not mediated by an action at the NMDA recognition site, because zinc had no effect on NMDA-displaceable L-[3H]glutamate binding. On the other hand, zinc dose-dependently inhibited [3H]glycine binding by a noncompetitive interaction. Stoichiometric analysis of equilibrium binding data indicated the presence of two [3H]glycine binding sites/[3H]TCP binding site. Comparison of the potencies of zinc in inhibiting glycine-dependent [3H]TCP association and [3H]glycine binding suggests that blockade of only one of the two glycine sites is sufficient to prevent [3H]TCP association. We hypothesize that synaptically released zinc inhibits NMDA receptor-mediated responses by binding to a site on the receptor/channel complex, reducing glycine binding, and thereby decreasing what would otherwise be a tonically present action of endogenous extracellular glycine.

Animals↗

[Application of tricalcium phosphate in one visit root canal treatment].

This paper reports the successful use of tricalcium phosphate (TCP) as periapical barrier material in animal experiments and clinics. The results of animal experiments showed that TCP had good biocompatibility with periapical tissues. At 6 weeks there were osteoids deposited on the surface of TCP. At 12 weeks osteogenesis occurred around the TCP particles and combined each other closely. At 24 weeks around the periapex there were bone deposits and a tendency of apical closure. On the basis of experiments TCP was used to treat 17 chronic periapicitis in 15 patients. All cases were young permanent teeth with immature roots. It is suggested that TCP can be applied directly in chronic periapicitis with open apices of young permanent teeth. From 6 to 12 months after filling with TCP the X-rays showed that radiolucencies reduced or eliminated, bone trabecula formed and periapical lamina dura reestablished.

Adolescent↗

N-methyl-D-aspartate receptor regulation of uncompetitive antagonist binding in rat brain membranes: kinetic analysis.

N-Methyl-D-aspartate (NMDA) receptor ligands regulate the binding of uncompetitive antagonists in membranes prepared from rat brain. To determine the mechanism of this regulation, we examined the kinetics of the binding of the radiolabeled uncompetitive antagonist [3H]N-(1-[thienyl]cyclohexyl) piperidine (TCP). Increasing concentrations of NMDA receptor agonists produced dose-dependent increases in the association and dissociation rate constants of TCP. The NMDA receptor antagonist amino phosphono valeric acid virtually abolished both the association and dissociation of TCP. Linear regression analysis detected a significant (p less than 0.001) correlation between the effect of NMDA receptor ligands on the apparent association and dissociation rate constants. The most parsimonious explanation of the data is that NMDA receptor ligands regulate TCP binding by controlling access of TCP to a transiently accessible or "guarded" binding site located in the receptor-coupled ion channel. An increase in affinity or number of TCP binding sites is neither necessary nor sufficient to explain the potentiation of TCP binding produced by NMDA agonists. This finding validates the use of uncompetitive antagonist binding as a measure of the functional activation of the NMDA receptor-coupled ion channel in isolated membrane preparations.

Animals↗

Enhanced stabilization of porous-coated metal implants with tricalcium phosphate granules.

Tricalcium phosphate (TCP) granules were tested as a possible alternative to bone graft as an interposition material to stabilize porous-coated metal implants in overreamed femoral intramedullary canals in dogs in vitro and in vivo. TCP granules provided as much stability as bone graft or a press fit in the in vitro testing. At six weeks in the in vivo system, the TCP stabilized specimens had an increased average resistance to tensile testing, but the increase was not significant. At six months, however, TCP-stabilized specimens exhibited a significant increase in resistance to pull-out force (p less than 0.05). Histologic examination revealed incorporation of the TCP granules in bone bridging from the endosteal cortex to the porous surfaces. TCP granules might be useful as a synthetic interposition material for stabilization of porous-coated metal components. TCP granules exhibited osteoconductive properties.

Animals↗

Resorption of, and bone formation from, new beta-tricalcium phosphate-monocalcium phosphate cements: an in vivo study.

Hard cylinders (4.7 x 10 mm) of two kinds of beta-tricalcium phosphate-monocalcium phosphate monohydrate-calcium sulfate hemihydrate (beta-TCP-MCPM-CSH) cements with and without beta-TCP granules (500-1000 microns) were implanted into holes drilled in rabbit femoral condyles for up to 16 weeks. Empty cavities were used as control. Cement resorption and new bone formation in the cylinders were evaluated with contact microradiography and quantified through an automatic image analysis system. At 4 weeks, both kinds of cement cylinders were surrounded by new bone. At 8 weeks, except for beta-TCP granules, both cement cylinders were almost completely resorbed and replaced by bone tissue. At 16 weeks the bone in the cavities of both cements recovered a trabecular pattern, but only the bone trabeculae in the initial cavity of the cement with beta-TCP granules became thick and mature. However, the cavities of the empty control were still empty and large. These results show that the beta-TCP-MCPM-CSH cements stimulate bone formation and are rapidly replaced by bone tissue. When added with nonresorbable beta-TCP granules, this cement maintains bone formation for a longer time.

Animals↗

Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts.

The chemical and topographic effects of commercially pure titanium on cell morphology and the regulation of matrix metalloproteinase-2 (MMP-2) gene expression, synthesis, and activity were investigated in early passage human gingival fibroblasts. Scanning electron microscopy showed that on smooth titanium (Ti), fibroblasts remained well spread and randomly oriented throughout the culture period. In contrast, cells on V-shaped grooved titanium (VTi) were oriented along the grooves by 16 h and proliferated in this organization throughout the culture period. The effects of substratum surface chemistry on MMP-2 expression were found to be distinct from those of topography. Northern hybridization analysis of fibroblasts cultured on Ti revealed an MMP-2 mRNA time-course expression pattern parallel to that observed on the tissue culture plastic (TCP) dishes, but at significantly lower levels at each time-point. The Ti mRNA levels were decreased by 34% at 16 h, 55% at 40 h, and 45% at 90 h relative to TCP. In contrast, MMP-2 mRNA expression on VTi showed both an altered time-course expression pattern and altered levels compared to Ti and TCP. Relative to TCP, VTi MMP-2 mRNA levels were approximately 80% less at 16 h and approximately 50% less at 40 h, but not significantly different at 90 h. Relative to Ti, VTi MMP-2 mRNA levels were approximately 75% less at 16 h, but approximately 40% greater at 40 h and approximately 70% greater at 90 h. These differences may be explained in part by the observed changes in MMP-2 mRNA half-life which decreased by approximately 40% on Ti but increased by over fourfold on VTi relative to TCP. The smooth Ti also showed an approximate twofold increase of MMP-2 secretion in the late cultures over TCP controls. These results indicate that substratum surface chemistry and topography-induced changes in cell shape can alter MMP-2 expression in normal fibroblasts. The molecular approach to investigating the major molecules involved in tissue degradation may provide sensitive indicators of tissue remodeling at the tissue-biomaterial interface.

Biocompatible Materials↗

Comparative bone growth behavior in granules of bioceramic materials of various sizes.

Various bioceramic materials were implanted into 6-mm-diameter holes made in the femoral condyles of mature Japanese white rabbits using different-sized granules to find an optimal material and granule diameter for use as a bone graft. Bioceramics include a bioinert ceramic (Alumina), surface-bioactive ceramics [hydroxyapatite (HAp) and Bioglass(R)], and resorbable bioactive ceramics [alphatricalcium phosphate (alpha-TCP), beta-TCP, tetracalcium phosphate (TeCP), Te. DCPD, Te. DCPA, and low-crystalline HAp]. Granule sizes were 100-300, 10, and 1-3 microm. Bone growth behavior varied with the kind of bioceramic and the size used. For surface-bioactive ceramics, 45S5 Bioglass(R) led to more rapid bone proliferation than synthetic HAp. In resorbable bioactive ceramics, the order of resorption was: low-crystalline HAp and OCP > TeCP, Te DCPD, Te DCPA > alpha-TCP, beta-TCP. In terms of biocompatibility, alpha-TCP was better than beta-TCP.

Aluminum Oxide↗

Transformation of biphasic calcium phosphate ceramics in vivo: ultrastructural and physicochemical characterization.

Specially prepared biphasic calcium phosphate (BCP) macroporous ceramics consisting of an intimate association of beta tricalcium phosphate (beta-TCP) and hydroxyapatite (HA) with beta-TCP/HA weight ratios of 15/85, 35/65, and 85/15 were implanted in surgically created periodontal osseous defects in dogs and recovered after 6 months. A decrease in average size of crystals in BCP ceramics and an increase in the size of microporosities in the surface and at the core of the ceramic after implantation were observed, indicating that in vivo dissolution has taken place. The resorbability (reflecting in vivo dissolution) of BCP ceramics depended on their beta-TCP/HA ratios, the higher the ratio, the greater the resorbability. The formation of microcrystals with crystallographic properties and Ca/P ratio similar to those of bone apatite crystals were also observed. The abundance of these crystals were directly related to the beta-TCP/HA ratio of the BCP ceramic before implantation. The formation of the bone apatite-like crystals may be due to the precipitation of calcium and phosphate ions released from the dissolving ceramic crystals (the beta-TCP component dissolving preferentially to the HA component). Results from this study suggested that one of the means of controlling resorbability (in vivo dissolution) of BCP ceramic is by varying its beta-TCP/HA ratio.

Animals↗

Dissolution control and cellular responses of calcium phosphate coatings on zirconia porous scaffold.

Different types of calcium phosphates [hydroxyapatite (HA), fluorapatite (FA), tricalcium phosphate (TCP), and their composites (HA + FA, HA + TCP)] were coated on a zirconia (ZrO(2)) porous scaffold using a powder slurry method. The ZrO(2) porous scaffold was intended for a load-bearing implant, and the apatite layers were coated to improve osteoconductivity. The insertion of an FA intermediate layer between the coating layer and ZrO(2) scaffold effectively suppressed the reaction between the calcium phosphate and ZrO(2) and maintained the coating layer at the initial powder composition. The obtained coating layer, of a thickness of approximately 30 microm, was relatively microporous and firmly adherent to the ZrO(2) scaffold. Dissolution tests in physiological solution showed typical differences depending on the coating layers, with the dissolution rate increasing in the order TCP > HA + TCP > HA > HA + FA > FA. This result suggests the functional coating of the calcium phosphates in view of tailoring the solubility. Osteoblast-like cells, MG63 and HOS, responded similarly in terms of cell growth, morphology, and proliferation rate regardless of the coating types, indicating favorable and comparable cell viability. However, the alkaline phosphatase (ALP) activity of the cells on the pure HA and HA composite coatings (HA + FA and HA + TCP) expressed at higher levels compared to those on pure FA and pure TCP coatings for both MG63 and HOS cells, suggesting a selective cell activity depending on the coating types. All the calcium phosphate-coated-ZrO(2) scaffolds showed higher ALP levels compared to pure ZrO(2) scaffold.

Biodegradation, Environmental↗

Effect of reinforcement particle size on in vitro behavior of beta-tricalcium phosphate-reinforced high-density polyethylene: a novel orthopedic composite.

Beta-tricalcium phosphate-reinforced high-density polyethylene (beta-TCP/HDPE) is a new biomaterial, which was made to simulate bone composition and study its capacity to act like bony tissues. This material was produced by replacing mineral component and collagen soft tissue of bone with beta-TCP and HDPE, respectively. The biocompatibility of composite samples with different volume fractions of TCP (20, 30, and 40 vol %) and two different particle sizes (80-100 and 120-140 mesh size) was examined in vitro using the osteoblast cell line G-292 by proliferation, alkaline phosphatase (ALP) production, and cell adhesion assays. Cell-material interaction on the surface of the composites was observed by scanning electron microscopy (SEM). The effect of beta-TCP particle size on behavior of the osteoblast cell line was compared between two groups of the composite samples containing smaller and larger reinforcement particle sizes as well as with those of a negative control. In general, results showed that the composite samples containing larger particles supported a higher rate of proliferation and ALP production by osteoblast cells after 3, 7, and 14 days of incubation compared to the composite samples with smaller particle size and control. Furthermore, more cells were attached to the surface of composite samples containing larger particle size when compared to the smaller particle size composites (p<0.05). This number was nearly equal with numbers adhered on negative control [tissue culture polystyrene (TPS)] and significantly higher in comparison with composite control [polyethylene (PE)] (p<0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division. These findings indicate that beta-TCP/HDPE composites are biocompatible, nontoxic, and in some cases, act to stimulate proliferation of the cells, ALP production, and cell adhesion when compared to the control counterparts. Furthermore, beta-TCP/HDPE samples with larger reinforcement particle size were shown to possess better biological properties.

Bone Substitutes↗

Rheological enhancement of mechanically activated alpha-tricalcium phosphate cements.

Most biocements are two- or three-component acid-based systems with large differences in the component particle sizes, which occurs by virtue of the differing processing routes. This work aimed to improve injectability and strength of a single reactive component cement, that is, mechanically activated alpha-tricalcium phosphate (TCP)-based cement by adding 13-33 wt % of several fine-particle-sized (d(50) of 0.5-1.1 microm) fillers [dicalcium phosphate anhydrous (DCPA), titanium dioxide (TiO(2)), and calcium carbonate] to the monomodal alpha-TCP matrix (d(50) = 9.8 microm). A high zeta-potential was measured for all particles in trisodium citrate solution. The fraction of alpha-TCP cement "injected" through an 800-microm hypodermic needle was found to be only 35% at a powder-to-liquid ratio of 3.5 g/mL. In contrast, the use of fillers decreased cement viscosity to a point, where complete injectability could be obtained. Mechanistically, these additives disrupted alpha-TCP particle packing yet decreased the interparticle spacing by a factor of approximately 5.5 such that the electrostatic repulsion effect was enhanced. A strength improvement was found when DCPA and TiO(2) were used as fillers despite the lower degree of conversion of these cements. Compressive strengths of precompacted cement samples increased from 70 MPa for unfilled alpha-TCP cement to 140 (110) MPa for 23 wt % DCPA (or TiO(2)) fillers as a result of porosity reduction. Strength improvement for more clinically relevant uncompacted cements was achieved by higher powder-to-liquid ratio mixes for filled cements such that maximum strengths of 90 MPa were obtained for 23 wt % DCPA filler compared with 50 MPa for single-component alpha-TCP cement.

Biocompatible Materials↗

Removal of surface by-products from sintered hydroxyapatite: effect of a chelation treatment on fibronectin adsorption and cell adhesion.

It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fibronectin's known affinity for calcium and the composition of the ceramic itself suggested that calcium release could be the main cause of this aggregation effect. It was then decided to investigate the effect of a surface chelation treatment on fibronectin adsorption, and MG63 cell adhesion, onto porous ceramics of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), and HA/TCP biphasic material (BCP). Those ceramics were immersed in an EDTA solution and the effect of this treatment on the material composition was assayed. X-ray diffraction data showed the presence of alpha- and beta-TCP phases in HA and BCP materials, which were both completely removed by the chelation treatment in the case of HA. On BCP, alpha-TCP was removed and beta-TCP partially dissolved. The TCP material, which was pure beta-TCP, underwent a mass loss, but no change in composition was observed. Adhesion of MG63 cells was overall higher on the fibronectin-coated EDTA-treated HA material, but was especially enhanced on EDTA-treated HA. Changes in surface morphologies, as compared with the use of scanning electron microscopy, did not seem to be related to the effects observed. The EDTA treatment proved to be a very efficient way of removing by-products of HA sintered materials, and thus enhancing the biocompatibility of the material.

Adsorption↗

Material characterization and in vivo behavior of silicon substituted alpha-tricalcium phosphate cement.

The possibility and biological effects of substituting silicon in alpha-tricalcium phosphate (alpha-TCP) by way of solid-state reaction have been evaluated. alpha-TCP powders with varying substitution amounts (1 and 5 mol % Ca2SiO4) were synthesized by reacting mixtures of CaCO3, Ca2P2O7, and SiO2, at a rate of 4 degrees C(min)(-1) to 1100 degrees C, left to dwell for 2 h and then heated to 1325 degrees C at 4 degrees C(min)(-1) and left to dwell for a period of 4 h. The powders were then rapidly quenched in air. Si incorporation could be verified by X-ray diffraction analysis, indicating an increase of the lattice volume with increasing Si content from 4284.1(8) to 4334(1) A3 for pure alpha-TCP and alpha-Si5%TCP, respectively. The hydrolysis of milled alpha-SiTCP powders was monitored by isothermal calorimetry, and the compressive strength of set cements was tested. The results showed changes in speed and amount of heat released during reactivity tests and a decrease in mechanical strength (60, 50, and 5 MPa) with increasing Si content. In vitro bioactivity of the set cements after soaking in simulated body fluid for 4 weeks was also tested. The formation of a bonelike apatite layer on the surface of the set cements could be observed and was thickest for 1%Si (20 microm). These results were in good agreement with the in vivo studies performed, which showed strong evidence that the cement containing 1% silicon doped alpha-TCP enhanced mesenchymal cell differentiation and increased osteoblast activity compared with alpha-TCP.

Animals↗