A biological study establishing the endotoxin limit of biomaterials for bone regeneration in cranial and femoral implantation of rats.

The purpose of this study was to accurately quantify the risk of endotoxin contamination in biomaterials for bone regeneration in order to establish the acceptable endotoxin limit. Collagen sheets containing varying amounts of purified endotoxin from Escherichia coli and dried, heat-killed E. coli or Staphylococcus aureus cells were implanted into cranial or femoral defects in rats. These defects were artificially prepared to a size of 5 × 5 mm or a diameter of 1 mm, respectively. The degree of osteoanagenesis was assessed by soft X-ray radiography and histopathology at 1 and 4 weeks after implantation. The collagen sheet containing the dried E. coli cells showed a dose-dependent delay in cranial and/or femoral osteoanagenesis at endotoxin activities of more than 33.6 EU/mg, at which no inflammatory response was observed. In contrast, no such observation occurred with the collagen sheet containing S. aureus cells. These results suggest that endotoxins may affect the process of osteoanagenesis. Additionally, the no-observed-adverse-effect level was 9.6 EU/mg, corresponding to 255 EU/kg body weight in rats. Interestingly, no delay in osteoanagenesis was induced by the implantation of collagen sheets containing purified endotoxin at any dose tested. This suggested that pure endotoxin implanted into tissues having poor circulation of bodily fluids without bleeding may not be recognized as a foreign substance and may not induce a significant biological response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1514-1524, 2017.

Protective effects of (6R)-5,6,7,8-tetrahydro-l-biopterin on local ischemia/reperfusion-induced suppression of reactive hyperemia in rat gingiva.

We herein investigated the regulatory mechanism in the circulation responsible for rat gingival reactive hyperemia (RH) associated with ischemia/reperfusion (I/R). RH was analyzed using a laser Doppler flowmeter. RH and I/R were elicited by gingival compression and release with a laser Doppler probe. RH increased in a time-dependent manner when the duration of compression was between 30 s and 20 min. This increase was significantly suppressed by N (ω)-nitro-l-arginine-methyl-ester (l-NAME), 7-nitroindazole (7-NI), and 2,4-diamino-6-hydroxypyrimidine (DAHP). However, RH was markedly inhibited following 60 min of compression. This inhibition was significantly decreased by treatments with superoxide dismutase (SOD), (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), and sepiapterin. The luminescent intensity of superoxide anion (O2 (•-))-induced 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazine-3-one (MCLA) was markedly decreased by SOD and BH4, but only slightly by sepiapterin. BH4 significantly decreased O2 (•-) scavenging activity in a time-dependent manner. These results suggested that nitric oxide (NO) secreted by the nitrergic nerve played a role in regulating local circulation in rat gingiva. This NO-related regulation of local circulation was temporarily inhibited in the gingiva by the I/R treatment. The decrease observed in the production of NO, which was caused by suppression of NO synthase (NOS) activity subsequent to depletion of the NOS co-factor BH4 by O2 (•-), played a partial role in this inhibition.

Contribution of nitrergic nerve in canine gingival reactive hyperemia.

Reactive hyperemia reflects a compensatory vasodilation response of the local vasculature in ischemic tissue. The purpose of this study is to clarify the mechanism of regulation of this response in gingival circulation by using pharmacological analysis of reactive hyperemia and histochemical analysis of gingival tissue. Application of pressure to the gingiva was used to create temporary ischemia, and gingival blood flow was measured after pressure release. Reactive hyperemia increased in proportion to the duration of pressure. Systemic hemodynamics remained unaffected by the stimulus; therefore, the gingival reactive hyperemia reflected a local adjustment in circulation. Gingival reactive hyperemia was significantly suppressed by nitric oxide (NO) synthase inhibitors, especially the neural NO synthase-selective antagonist 7-nitroindazole, but not by anticholinergic drugs, ß-blockers, or antihistaminergic drugs. Moreover, immunohistochemical staining for neural NO synthase and histochemical staining for NADPH diaphorase activity were both positive in the gingival perivascular region. These histochemical and pharmacological analyses show that reactive hyperemia following pressure release is mediated by NO-induced vasodilation. Furthermore, histochemical analysis strongly suggests that NO originates from nitrergic nerves. Therefore, NO may play an important role in the neural regulation of local circulation in gingival tissue ischemia.

Reactive oxygen species scavenging activity of Jixueteng evaluated by electron spin resonance (ESR) and photon emission.

Jixueteng, the dried stem of Spatholobus suberectus Dunn (Leguminosae), is a traditional Chinese herbal medicine that is commonly classified as a herb that promotes blood circulation and can be used to treat blood stasis. The aim of this study was to examine the reactive oxygen species (ROS) scavenging activity of Jixueteng and other herbal medicines. The ROS scavenging activities of the water extracts of Jixueteng, Cnidium officinale and Salvia miltiorrhiza were examined using an electron spin resonance (ESR) technique and faint luminescence measurement. The ESR signal intensities of the superoxide anion (O2·) and hydroxyl radical (HO·) were reduced more by Jixueteng than the other herbal medicines we tested. High photon emission intensity to hydrogen peroxide (H202) and HO· was observed in Jixueteng using the XYZ chemiluminescence system that was used as faint luminescence measurement and analysis. The results of the present study revealed that the ROS scavenging activity of 8% Jixueteng was the strongest among the herbal medicines we tested. It has been reported that Jixueteng includes various polyphenols. In the ROS scavenging activity by Jixueteng, it is supposed that the antioxidant activity caused by these polyphenols would contribute greatly. In conclusion, a water extract component of Jixueteng had potent free radical scavenging activity and an antioxidative effect that inhibited the oxidative actions of O2·â», H2O2 and HO·. Therefore, Jixueteng represents a promising therapeutic drug for reactive oxygen-associated pathologies.

Gingival vascular functions are altered in type 2 diabetes mellitus model and/or periodontitis model.

The association of vascular reactivity between diabetes and periodontal disease has not been clarified. Gingival blood flow was measured by laser Doppler flowmetry for 31 weeks in Wistar rats, Wistar rats orally challenged with Porphyromonas gingivalis (Wistar rats + Porphyromonas gingivalis), Goto-Kakizaki rats, and Goto-Kakizaki rats orally challenged with Porphyromonas gingivalis (Goto-Kakizaki rats + Porphyromonas gingivalis). Effects of alveolar bone resorption on periodontal tissue was enhanced in Wistar rats + Porphyromonas gingivalis, and Goto-Kakizaki rats, with this effect being significantly enhanced by Goto-Kakizaki rats + Porphyromonas gingivalis. Using the L-band electron spin resonance technique, we succeeded in measuring oxidative stress as decay rate constant (K(1) and K(2)) of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy in the oral and maxillofacial region of the animal models. The decay rate constant (K(1)) of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy was significantly greater in the oral and maxillofacial region of Goto-Kakizaki rats + Porphyromonas gingivalis compared to Wistar rats, Wistar rats + Porphyromonas gingivalis and Goto-Kakizaki rats groups. Gingival reactive hyperemia was attenuated by periodontal disease, and this effect was also remarkable in the diabetes mellitus model. Taken together, we found that vascular endothelial function was decreased in diabetes mellitus and/or periodontal disease animal models due to increasing oxidative stress in the gingival circulation.

Inhibitory effects of Jixueteng on P. gingivalis-induced bone loss and osteoclast differentiation.

OBJECTIVE: The aim of this study was to investigate the possibility of Jixueteng as a preventive and therapeutic drug for the periodontitis. We investigated the inhibitory effects of Jixueteng on Porphyromonas gingivalis-induced bone loss in mice, antibacterial activity against P. gingivalis and differentiation of osteoclast and viability of cells. MATERIALS AND METHODS: Fifty-four male, 4-week-old C57BL/6N mice, were randomly divided into the following three groups of 18 mice each; group A served as the P. gingivalis non-infected control (sham group), group B was infected orally with P. gingivalis and group C was administered Jixueteng extract in drinking water and was then infected with P. gingivalis. In order to evaluate the effect of Jixueteng, the distance from the alveolar bone crest to the cemento-enamel junction was determined. P. gingivalis suspension was exposed for 1, 15 and 60 min to 5 ml of the Jixueteng extract. Furthermore, to clarify the mechanism of the inhibitory effects of Jixueteng on osteoclast formation, Jixueteng extract was added to the culture of mouse bone marrow cells, osteoclast precursor. RESULTS: Administration of Jixueteng along with P. gingivalis infection significantly reduced alveolar bone loss compared to P. gingivalis infection. Jixueteng treatment at the concentration of 0.01% significantly inhibited osteoclast formation. The addition of Jixueteng extract (0.1%, 0.01%, and 0.001%) to the culture showed a significant inhibition of the number of surviving osteoclasts in a dose-dependent manner. CONCLUSION: Jixueteng has an antibacterial activity against P. gingivalis and inhibitory effects on osteoclastogenesis, it may be useful as a therapeutic drug in the treatment of P. gingivalis-induced periodontitis.

Co-treatment with basic fibroblast growth factor and 17beta-estradiol in the presence of dexamethasone accelerates bone formation by rat bone marrow stromal cell culture.

PURPOSE: Bone marrow stromal cells (BMSCs) are a promising cell source in applications for tissue engineering and regenerative medicine. Optimization and control of the growth and differentiation of cultivated cells can be achieved by the administration of growth factors and hormones in vitro. This study provided experimental information on the enhancement of the osteogenic potential of rat BMSCs in vitro and in vivo. METHODS: Mineralized nodule formation of rat BMSCs in culture for 3 weeks with dexamethasone (Dex)-treated media supplemented with both basic fibroblast growth factor (bFGF) and 17beta -estradiol (E2) was examined by histology. In porous beta-tricalcium phosphate (beta - TCP), proliferation, migration, and differentiation of BMSCs were examined by histology and transmission electron microscopy. After culturing, the composites were subcutaneously implanted into syngeneic rats. The tissues with implants were harvested after 4 weeks and evaluated microscopically by using histological stain. RESULTS: Dex-treated media supplemented with both bFGF and E2 was the most effective in mineralized nodule formation of BMSCs in vitro. Light and electron microscopy revealed the presence of many cells with developed rough endoplasmic reticulum. Bone formation in the BMSC/beta -TCP composites in cultures in vitro for 3 weeks was observed histologically at 4 weeks after implantation. When BMSC/beta -TCP composites were cultured in Dex-treated media supplemented with both bFGF and E2, the amount of bone formation at implants was substantially greater than that of composites cultured in Dex-treated media supplemented with bFGF. CONCLUSION: The combined use of bFGF and E2 could effectively improve the bone-forming ability of BMSCs.

Direct assessments of the antioxidant effects of propofol medium chain triglyceride/long chain triglyceride on the brain of stroke-prone spontaneously hypertensive rats using electron spin resonance spectroscopy.

BACKGROUND: Antioxidant anesthetics such as propofol (2,6-diisopropylphenol) directly inhibit lipid peroxidation via the generation of reactive oxygen species. Currently, there are no other studies regarding the direct effects of propofol medium chain triglyceride/long chain triglyceride (MCT/LCT) on reactive oxygen species generation or in experimental models of reactive oxygen species-induced oxidative stress in the brain. METHODS: The authors investigated the effects of propofol MCT/LCT on reactive oxygen species (hydroxyl radical or superoxide) by electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. The effects of propofol MCT/LCT on oxidative stress in the brain of Wistar-Kyoto rats or stroke-prone spontaneously hypertensive rats were investigated by using an in vivo L-band electron spin resonance system to monitor the decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl as a nitroxyl spin probe. RESULTS: These studies provided direct evidence that propofol MCT/LCT inhibited hydroxyl radical generation, but not superoxide generation. Regarding the hydroxyl radical from the Fenton system, it is likely to be due to the scavenging effects of vehicle. Anesthesia with propofol MCT/LCT reduced the degree of the high oxidative stress in the brain of stroke-prone spontaneously hypertensive rats. CONCLUSION: The current data show that propofol, mixed with clinical reagents (propofol MCT/LCT), resulted in the down-regulation of high oxidative stress due to scavenging hydroxyl radical, as demonstrated by in vitro or in vivo electron spin resonance analysis. These results led to reduced levels of hydroxyl radical, formed by brain injury such as stroke, and may therefore provide advantages for neuroprotection during anesthesia for craniotomy, e.g., in cases of brain disease.

Nitric oxide levels in rat hypothalamus are increased by restraint stress and decreased by biting.

Mastication, which includes biting, is of great importance not only for the intake of food but also for the mental, physical and physiological functioning of the body. For example, biting suppresses the stress response. Although biting and nitric oxide (NO) appear to modulate brain dynamics during stress, the underlying mechanisms have not been elucidated. In this study, we examined the effect of biting during restraint stress on NO levels in the rat hypothalamus. To this end, we used NO-selective electrodes that were calibrated by electron spin resonance (ESR) spectroscopy. We implanted the electrodes and probes for perfusion of solutions into the brain of rats, near the hypothalamus. Saline containing 10 mM N-nitro-L-arginine methyl ester (L-NAME), which is one of the most commonly used inhibitors of nitric oxide synthase (NOS), was employed as the perfusate. L-NAME prevented increases in NO levels in the rat hypothalamus that were induced by restraint stress and biting. Hypothalamic NO levels in rats under restraint stress for 180 min were increased above levels observed in unrestrained control rats. The increase in hypothalamic NO (from 2.123 muM to 4.760 muM) during restraint stress was reduced after biting for 30 min. The decay rate of NO levels after biting was -0.584 pA/min (-0.071 muM/min). We conclude that: (i) it is possible to evaluate NO levels in vivo in rat brain; (ii) NO levels are increased by restraint stress; and (iii) this increase is prevented by biting behavior.

Real-time monitoring of nitric oxide in ischemic myocardium using an NO-selective electrode calibrated by electron spin resonance.

Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.

Endothelium-dependent vasorelaxation induced by black currant concentrate in rat thoracic aorta.

We investigated the effect of black currant (BC) concentrate on smooth muscle in rat thoracic aorta. BC concentrate dose-dependently relaxed the norepinephrine (0.1 microM)-precontracted aorta, and the response was abolished after endothelium removal. Both oxyhemoglobin (1 microM), a nitric oxide (NO) scavenger, and IH-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 0.5 microM), an inhibitor of guanylyl cyclase (GC), inhibited the relaxing effect of BC concentrate. NG-nitro-L-arginine methyl ester (L-NAME, 10 microM), a nitric oxide synthase (NOS) inhibitor, inhibited the relaxation, and the subsequent addition of L-arginine (1 mM), a NOS substrate, reversed the inhibitory effects of L-NAME. Neither indomethacin (10 microM), an inhibitor of cyclooxygenase, nor atropine (1 microM), an antagonist of muscarinic receptors, modified the effect of BC concentrate. Diphenhydramine (3 microM) and chlorpheniramine (2 microM), selective antagonists of H1-receptors, inhibited the relaxation, but cimetidine (0.3 mM), a selective antagonist of H2-receptors, did not affect the relaxation. These results indicate that, in the rat aorta, BC concentrate enhances synthesis of NO, which subsequently induces the endothelium-dependent vasorelaxation via the H1-receptors on the endothelium.

Role of nitric oxide in post-ischemic gingival hyperemia in anesthetized dogs.

The possible involvement of nitric oxide (*NO) in the preservation of blood flow to the canine gingiva after compression of gingival tissue was studied. Gingival blood flow, gingival tissue oxygen partial pressure (PO2), external carotid arterial blood pressure and external carotid arterial blood flow were monitored before, during, and after compression of gingival tissue in the presence and absence of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine-methyl-ester (L-NAME). Compression of gingival tissue resulted in an immediate decrease in gingival blood flow and tissue PO2. After the compression of gingival tissue, hyperemia was observed in the gingiva, which depended on the duration of ischemia. Gingival tissue PO2 slowly recovered during hyperemia. Pretreatment with L-NAME (60 mg/kg, i.a.) significantly suppressed reactive hyperemia in gingival tissue. The L-NAME-suppressed reactive hyperemia was partially reversed by treatment with L-arginine (60 mg/kg, i.a.). In addition, *NO was detected using an *NO selective electrode during interruption of blood flow and during reactive hyperemia in the gingiva. These results suggest that *NO contributes to the vasodilation during reactive hyperemia in gingival tissue, and aids in the maintenance of homeostasis in gingival circulation.