Alveolar bone regeneration using absorbable poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblast growth factor.

The aim of this study was to evaluate the effects of combining a porous poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblastic growth factor (bFGF) on bone regeneration in mandibular ridges. Four full-thickness saddle-type defects (10 mm long x 5 mm deep) were symmetrically created in both edentulous mandibular alveolar ridges of 6 beagles. The dome-shaped membrane was secured to each defect site, and a gelatin sponge containing 200 microg bFGF was implanted on the left side of each defect (experimental group). Only the membranes (control group) were secured to the defect sites on the right. Three and 6 months later, 3 animals were killed. Bone regeneration was analyzed by soft X-ray photographs, micro-computed tomography (CT) images, and peripheral quantitative CT (pQCT), and then examined histologically. Soft X-ray examination revealed an increase in new bone volume in the experimental group 6 months postoperatively. pQCT showed that immature bone density was higher in the experimental group. Micro-CT images revealed well formed new bone along the original contour of the dome-shaped membrane in the experimental group. Histologically, inflammatory infiltration of tissue surrounding the membranes was slight. These results suggest that combining the poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and bFGF-gelatin sponge is promising for alveolar ridge reconstruction.

Characterization by electron spin resonance spectroscopy of reactive oxygen species generated by titanium dioxide and hydrogen peroxide.

The influence of reactive oxygen species (ROS) on the surface modification of titanium implants and osseointegration is unclear. The aim of this study was to evaluate the ability of titanium dioxide (TiO2) to generate ROS in the presence of H2O2 and to determine whether any ROS thus generated play a role in osseointegration, as measured by electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrolline-N-oxide (DMPO). We demonstrate that TiO2 together with H2O2 generated hydroxyl radicals (HO*), as shown by a time-dependent increase in the spin concentration of the ESR signal for the DMPO-OH spin adduct, indicating HO* generation. Interestingly, irradiated TiO2 with H2O2 generated the superoxide (O2*-), as shown by an increase in the spin concentration of the signal for the DMPO-OOH spin adduct, indicating O2*- generation during the period of irradiation (0-5 min). These results suggest that ROS generated from the TiO2 layer may be involved in creating appropriate conditions for the osseointegration of dental implants into alveolar bone tissues.

[Microcirculatory hemodynamics in oral tissues with reference to neurogenic response and reactive oxygen species interaction].

The primary purpose of the microcirculation is to transport nutrients and oxygen and to remove metabolic waste products from tissues. It is also well known that the fundamental mechanism for vascular control is the local regulation of the basal vascular tone, which is reinforced by blood pressure and counteracted by tissue metabolites. Thus, the well-being of the tissue depends on the circulatory transport process, which is governed by many functional parameters of the microcirculation such as blood flow, blood volume, intravascular and extravascular pressures, and capillary permeability. Inflammatory reactions in oral tissues can be initiated by many different insults to the tissues, and the reaction itself can be expressed in various ways. In addition, the tissues seem to have many "backup" systems, so that any one response can be produced in several ways, which is important for a reaction that has a survival value. A recent concept is that repeated stimulation of sensitive teeth may induce pulpal changes; this could occur through induction of neurogenic inflammation and alteration of pulpal blood flow. One possibility is that production of reactive oxygen species, as well as release of the sensory neuropeptides, at sites of inflammation contributes to alterations in local blood flow. In addition to the part played by the neurogenic mediators, nitric oxide participation and its interaction with oxygen-derived free radicals in oral tissue hemodynamics are also discussed.

Inhibition by hydroxyl radicals of calcitonin gene-related peptide-mediated neurogenic vasorelaxation in isolated canine lingual artery.

Canine lingual arteries are innervated by calcitonin gene-related peptide (CGRP)-containing vasodilator nerves. Although the vascular system might be considered as the first target of oxygen-derived free radicals in some of the pathophysiological conditions, the effect of oxygen-derived free radicals on neurotransmission in CGRP nerves remains unknown. We, therefore, investigated the role of oxygen-derived free radicals generated from Fenton's reagent (3 x 10(-4) M H2O2 plus 2 x 10(-4) M FeSO4) on CGRP-mediated neurogenic relaxation of canine lingual artery ring preparations. In all experiments, endothelium-denuded preparations (which were suspended in the tissue bath for isometric tension recordings) were treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. The periarterial nerve stimulation (10 V, 4-16 Hz, for 45 sec), exogenous CGRP (10(-8) M) or the ATP-sensitive K+ channel opener cromakalim (10(-6) M) produced relaxation of the rings at a stable plateau tension by the addition of norepinephrine (10(-5) M); the relaxations elicited by CGRP and cromakalim were human CGRP-(8-37)- and glibenclamide-abolishable, respectively. When the nerve stimulation, CGRP and cromakalim were given after H2O2/FeSO4 exposure (Fenton's reagent was removed from the tissue bath), the observed relaxations were markedly diminished. The effects afforded by the early exposure to H2O2/FeSO4 reaction of the preparations were significantly protected by catalase (100 U/ml, H2O2 scavenger), dimethylthiourea (1 mM, H2O2 and HO. scavenger), dimethyl sulfoxide (100 mM, HO. scavenger), deferoxamine (1 mM, a powerful iron chelator) and by a cocktail of catalase-deferoxamine. Generation of HO. from H2O2/FeSO4 was studied by electron spin resonance spectroscopy using the spintrap 5,5-dimethyl-1-pyrroline-N-oxide. We found that H2O2/ FeSO4 reaction formed a 1:2:2:1 quartet, characteristic of the HO-5,5-dimethyl-1-pyrroline-N-oxide spin adduct. After exposure to capsaicin (10(-5) M) or H2O2/FeSO4 of the artery ring preparations, the intensity of CGRP-like immunoreactivity of the periarterial nerves was reduced drastically; the relaxation caused by the nerve stimulation was nearly fully inhibited by capsaicin and H2O2/FeSO4 reaction. The relaxant response, however, to nitroglycerin (10(-5) M) in the presence of norepinephrine to induce tone was unaffected by the early H2O2/ FeSO4 exposure. The data obtained from the present study indicate that HO., rather than H2O2, is the active agent in CGRP-mediated neurogenic relaxation. It is suggested that the HO. can deplete endogenous CGRP localized prejunctionally and also damage CGRP-induced relaxation of canine lingual artery preparations that is caused by activation of ATP-sensitive K+ channels at postjunctional sites. It is also postulated that the second messenger system of the relaxation mediated, at least, by cyclic GMP may be less susceptible to HO..

[Measurement of reactive oxygen species in a biological system and its perspectives].

In recent years, reactive oxygen species have been implicated in the pathogenesis of a wide variety of disorders. Although the existence of reactive oxygen intermediates in drug metabolism can be inferred from end product analysis or from the effects of antioxidants or enzymes such as superoxide dismutase, only the technique of electron spin resonance (ESR) allows the direct detection of these highly reactive species. However, some free radical species cannot be detected by ESR due to their extremely short half-lives, which result in low steady-state concentrations of the radicals or to short radical relaxation times, which lead to a very broad line. These facts made recent development of spin-trapping and chemiluminescence techniques are widely used to detect free radicals. The goal of this paper is to introduce the various assays available for measurement of reactive oxygen species in biological models. This paper will focus on two topics: (1) the spin-trapping/ESR technique in vitro and vivo and (2) the chemiluminescence-optical biosensor application of this technique, a very sensitive method that has the advantage of being able to provide continuous, online, nondestructive monitoring of reactive oxygen species.

Calcitonin gene-related peptide mediated neurogenic vasorelaxation in the isolated canine lingual artery.

The nature of neurogenic relaxation was investigated in ring preparations of canine lingual artery. In all experiments, the preparations were previously treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. In the presence of norepinephrine (10(-5) M) to induce tone, electrical stimulation (10 V, 4 to 16 Hz, for 45 sec) produced relaxation of the rings in an endothelium-independent fashion. The relaxant response in endothelium-denuded rings was not changed by propranolol (10(-5) M), and atropine (10(-5) M) did not affect the relaxation elicited by electrical stimulation in endothelium-intact rings. NG-monomethyl-L-arginine (10(-4) M) or NG-nitro-L-arginine methyl ester (10(-4) M), a nitric oxide (NO) synthase inhibitor, had no effect on the electrical stimulation-induced relaxation of endothelium-denuded rings. Human calcitonin gene-related peptide (CGRP)-(8 - 37) (2 x 10(-8) M), a CGRP1-receptor antagonist, inhibited neurogenic relaxation of endothelium-denuded rings; substance P (10(-6) M) failed to mimic the observed effect of electrical stimulation. The demonstrated effect of electrical stimulation was inhibited by glibenclamide (10(-5) M), but not tetraethylammonium (2 x 10(-4) M); glibenclamide abolished the relaxation in response to exogenous CGRP or the ATP-sensitive K+ channel opener cromakalim (10(-6) M) in endothelium-denuded rings. Moreover, tetrodotoxin (3.13 x 10(-6) M) inhibited the relaxation of endothelium-denuded rings induced by electrical stimulation. The relaxation was selectively inhibited when endogenous CGRP had been depleted from perivascular nerves by capsaicin (10(-6) M). These results suggest that CGRP, but not NO, released from non-adrenergic non-cholinergic nerves by electrical stimulation produces relaxation of canine lingual artery that is mediated by activation of CGRP1 receptors.

[A mechanism underlying histamine-induced contraction in isolated rabbit lingual artery].

To understand the contractile response to histamine, drug-induced tension developments were measured in intact and staphylococcal alpha-toxin-treated permeabilized smooth muscle preparations of rabbit lingual artery. Histamine produced an endothelium-independent contraction; the observed effect was antagonized by diphenhydramine and was attenuated by nifedipine. Histamine produced only transient contraction in the Ca(2+)-free bathing media. In permeabilized preparations, histamine-induced contraction was abolished by ryanodine (in the presence of caffeine) or prior repeated application of caffeine; however, contraction produced by IP3 was still observed under the same experimental condition as that of the histamine study. Histamine- or caffeine-induced contraction was abolished in the permeabilized preparations by prior repeated application of IP3; caffeine or IP3 produced contraction after repeated application of histamine. Ryanodine in the presence of histamine was ineffective on caffeine-induced contraction, suggesting that histamine by itself may not have the ability to act on caffeine-sensitive Ca2+ release channels. Neomycin and H-7 completely abolished the histamine-induced contraction. Hence, it is suggested that histamine can contract the lingual artery via H1-receptor-coupled phospholipase C activation, and the contraction consists of a phasic response evoked by Ca2+ release from the IP3- and caffeine-sensitive Ca2+ storage site and the tonic response generated by the voltage-dependent influx of Ca2+. It is also postulated that IP3 produced by histamine may be able to produce Ca2+ release from only a part of the intracellular Ca2+ stores in the smooth muscle of rabbit lingual artery.

Oxygen free radical-mediated selective endothelial dysfunction in isolated coronary artery.

To understand the direct involvement of free radicals causing reduction in endothelium-dependent relaxation of isolated canine coronary ring preparations, this study was undertaken to examine the effect of free radicals generated from dihydroxy fumarate (DHF) plus Fe(3+)-ADP or from H2O2 plus FeSO4. The vasodilators (acetylcholine, bradykinin, A23187, and nitroglycerin) were given after DHF/Fe(3+)-ADP or H2O2/FeSO4 was removed from the organ chamber. The earlier DHF/Fe(3+)-ADP exposure produced an attenuation of the relaxation of the rings induced by acetylcholine, bradykinin, or A23187 but not of the relaxation induced by nitroglycerin. The observed effect of previous DHF/Fe(3+)-ADP exposure was significantly protected in the vessels isolated from the dogs treated with alpha-tocopherol. In the experiments for assessing the effect of various scavengers, 1O2 scavenger histidine or iron chelator deferoxamine effectively protected the attenuation induced by DHF/Fe(3+)-ADP exposure of the relaxation elicited by acetylcholine; superoxide dismutase (SOD), catalase, or dimethyl sulfoxide (DMSO) had no effect on this system. Furthermore, the relaxation elicited by acetylcholine, but not nitroglycerin, was significantly attenuated by the earlier exposure to .OH generated by Fenton's reagent (H2O2+FeSO4); the attenuation was significantly protected by DMSO. These results are consistent with the view that .OH, 1O2, and/or iron-dependent reactive species selectively damage endothelium-dependent relaxation as opposed to endothelium-independent relaxation in endothelium-intact coronary ring preparations. It is also postulated that lipid peroxidation may be responsible for this effect.

[The contractile response of isolated lingual arteries from rabbits treated with bacterial lipopolysaccharide via the stimulation of B1-receptors for kinins].

The effect of intravenous injection of 10 micrograms of a lipopolysaccharide (endotoxin) extracted from E. coli to rabbits on the responses of isolated lingual arteries to des-Arg9-bradykinin (a specific kinins B1-receptor agonist) was studied. Endotoxin injection led to the appearance of the endothelium-independent contractile effect of des-Arg9-bradykinin in the arteries; endotoxin elicited this response when administered at 1, 5 or 20 hr before the experiment, in a time-interval dependent manner. The contractile response to des-Arg9-bradykinin of the arteries isolated from animals receiving endotoxin 20 hr before the experiment was attenuated by des-Arg9-[Leu8]-bradykinin (a specific inhibitor of kinins B1-receptor) or pretreatment of the animals with an inhibitor of protein synthesis (cycloheximide and actinomycin D). When compared with the effect of des-Arg9-bradykinin, bradykinin (a potent kinin B2-receptor, but weak B1-receptor stimulant) caused slight contraction of the arteries; however, this effect was not endotoxin-dependent and was not modified by des-Arg9-[Leu8]-bradykinin. Effect of in vitro preincubation with endotoxin of the arteries isolated from animals receiving saline 20 hr before the experiment was further studied. The preincubation (for 1 and 5 hr) with endotoxin of the arteries in the presence or absence of plasma had no effect on the sensitivity of the arteries to des-Arg9-bradykinin; the sensitivity was also unaffected in the presence or absence of endotoxin, thus suggesting that there is no interaction between endotoxin and some plasma-related factors with the appearance of the contraction in response to the kinin B1-receptor agonist in the arteries in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct pharmacological action of vasoactive substances on pulpal blood flow: an analysis and critique.

An adequate blood supply to the dental pulp is essential to the health of the tooth; therefore, there have been a number of efforts to study pulpal blood flow and the factors which influence it. However, blood flow to the dental pulp is relatively inaccessible and apparently quite low. Consequently, it is difficult to obtain accurate flow measurements, partly owing to methodological difficulties with the small size of the tissue and its enclosure within rigid walls. In this study, the effects of locally applied vasoactive substances and their specific antagonists on pulpal blood flow have been examined by laser Doppler flowmetry. It is the purpose of this article to examine, in-depth, the involvement of endogenous vasoactive substances in the regulatory mechanism of blood flow within the dental pulp and expand our knowledge of pulpal microcirculatory hemodynamics.

[Effect of vasoactive substances and electrical stimulation of the inferior alveolar nerve on blood flow in the dental pulp in dogs].

The regulatory mechanism of the pulpal microcirculatory hemodynamics was investigated by measuring pulpal blood flow (PBF) in dogs by means of a laser doppler flowmeter. Application of vasodilators (acetylcholine, isoproterenol, histamine, bradykinin and substance P) to the prepared cavity caused an increase in PBF, and norepinephrine reduced PBF. These vasoactive substance-induced responses, but not the bradykinin-induced response, were inhibited by i.v. injection of antagonists (atropine, propranolol, diphenhydramine and [D-Pro2, D-Trp7,9]-substance P). The effect of bradykinin was inhibited by indomethacin, but not by des-Arg9-[Leu8]-bradykinin. Furthermore, prostaglandin E2 produced a concentration-dependent increase in PBF. These results suggest that acetylcholine, histamine, bradykinin, substance P and norepinephrine, if present, influence the local vasomotor regulation in the dental pulp, and that bradykinin may exert the effect via prostaglandin synthesis. Based on this suggestion, the effect of electrical stimulation of the distal end of the cut inferior alveolar nerve on PBF was studied. The nerve stimulation-induced increase in PBF was inhibited by indomethacin, but not by atropine, propranolol, diphenhydramine, soybean trypsin inhibitor, aprotinin, des-Arg9-[Leu8]-bradykinin, or by [D-Pro2, D-Trp7,9]-substance P. The experiments show that the increase in PBF produced by stimulation of the inferior alveolar nerve is not mediated by common efferent vasodilatory mechanisms, and it is probably mediated by prostaglandin release via the sensory nerve axon reflex mechanism.

Free radicals-induced changes in mesenteric microvascular dimensions in the anesthetized cat.

Effect of free radicals on microvascular dimensions was studied in anesthetized cat intestinal mesentery. Generation of free radicals by xanthine oxidase, acting on endogenous substrates (e.g., xanthine, hypoxanthine), produced sustained vasodilation at pH 7.4 or at pH 6.6. These effects were reversed by superoxide dismutase (SOD), a superoxide radical (.O2-) scavenger, at pH 7.4, and by SOD plus hydroxyl radical (.OH) scavenger d-mannitol at pH 6.6. These findings suggest that the sustained vasodilation is caused by .O2- and by .OH derived from .O2- at low pH.