Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

UNLABELLED: Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

Simvastatin enhances bone formation around titanium implants in rat tibiae.

Statins are cholesterol-lowering drugs that have been reported to promote bone formation. The purpose of this study was to investigate the effect of simvastatin on the enhancement of bone formation around titanium implants. Thirty-week-old female rats received pure titanium implants in both tibiae. The animals were intra-peritoneally administered 0, 0.125, 1, 5 or 10 mg kg(-1) of simvastatin daily. After 30 days, the animals were sacrificed, and specimens were prepared. The bone contact ratio of the implant, bone density in the medullary canal and percentage of cortical bone were obtained. Markers for bone turnover were also measured using sera collected at the time of euthanasia. In the medullary canal, a scanty amount of bone was observed in the 0, 0.125 and 1 mg kg(-1) groups. In contrast, in both the 5 and 10 mg kg(-1) groups, thicker bone trabeculae were abundant. Histometric observations showed that the bone contact ratio and the bone density of both groups were significantly greater than those of the other groups (anova, P < 0.01). However, no significant difference in the percentage of cortical bone was found between groups. Serum chemistry showed that statin increased bone formation markers and decreased bone resorption markers. In conclusion, although the dose equivalent to that used in human patients with hypercholesterolemia was not effective, a simvastatin dose of 5 mg kg(-1) or higher increased medullary bone formation around the titanium. In contrast, no effect of simvastatin on pre-existing cortical bone was indicated.

Platelet-rich plasma suppresses osteoclastogenesis by promoting the secretion of osteoprotegerin.

BACKGROUND AND OBJECTIVE: Platelet-rich plasma is characterized by containing fundamental protein growth factors. Although many in vitro studies have documented the capability of platelet-rich plasma to induce the growth of osteoblasts or osteoblast-like cells, the effect of platelet-rich plasma on osteoclastogenesis has not yet been studied. The aim of the present study was to evaluate the effects of platelet-rich plasma and platelet-poor plasma on osteoclastogenesis with rat bone marrow cell culture. MATERIAL AND METHODS: Platelet-rich plasma and platelet-poor plasma were produced from the whole blood of rat. For cell culture, rat bone marrow cells were isolated from rat tibiae and then treated with 1,25alpha dihydroxy vitamin D(3) and with different concentrations of platelet-rich plasma or platelet-poor plasma. After 4 d of culture, rat bone marrow cells were stained with tartrate-resistant acid phosphatase (TRAP), and TRAP-positive cells that had more than three nuclei (TRAP-positive multinucleated cells) were counted as osteoclast-like cells. Osteoprotegerin, known as an osteoclastogenesis-related factor, cells was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Although platelet-poor plasma had no effect on the formation of TRAP-positive multinucleated cells, platelet-rich plasma decreased the number of TRAP-positive multinucleated cells in a dose-dependent manner. The amount of osteoprotegerin produced from rat bone marrow cells and from MC3T3-E1 cells was enhanced in platelet-rich plasma-treated groups. CONCLUSION: Under our experimental conditions, platelet-rich plasma decreased the formation of TRAP-positive multinucleated cells and increased the secretion of osteoprotegerin. This study suggests that platelet-rich plasma suppresses osteoclastogenesis, therefore inhibiting bone resorption. In addition we also demonstrated that platelet-rich plasma increased the secretion of osteoprotegerin, an inhibitor for osteoclast formation, thus suggesting that the enhancement of osteoprotegerin secretion induces this inhibitory effect.

Distribution of new methylene blue injected into the caudal epidural space in cattle.

To investigate the distribution of solutions injected into the first intercoccygeal epidural space, 24 adult, standing cattle were randomly assigned to 5-, 10- and 20-mL groups and injected with 0.12% new methylene blue (NMB) in 0.9% saline. Ten heifers received 1 mL NMB solution/100 kg of body weight. There was a significant correlation between the injected volume and the number of cranially stained spinal segments in three adult cattle groups (correlation coefficient R2=0.46; P<0.0001). In three cattle, NMB solution did not distribute more than one spinal segment cranially from the injection site due either to fibrosis of the epidural tissue or to inadvertent intravenous administration into the epidural vein. The study showed that the larger the volume of solution injected, the greater the spread with increased individual variation. The results could form the basis for determining the volume of injection required and for evaluating the pharmacokinetics of anaesthetics used in caudal epidural anaesthesia.

Comparison of xylazine, lidocaine and the two drugs combined for modified dorsolumbar epidural anaesthesia in cattle.

Twenty-four Holstein cattle scheduled for flank surgery in a standing position were randomly assigned to four groups of six. A 16 G, 120 mm Tuohy needle was inserted into the first interlumbar epidural space and its position was confirmed by the hanging drop technique. After air had been allowed to enter freely for approximately one minute, the epidural needle was slowly inserted 7 to 10 mm deeper to penetrate the epidural fat, and anaesthetic solution containing either 0.05 mg/kg bodyweight xylazine hydrochloride (xylazine), 0.025 mg/kg xylazine, 0.025 mg/kg xylazine and 0.1 mg/kg lidocaine hydrochloride (lidocaine), or 0.2 mg/kg lidocaine alone was administered. Signs of sedation were observed in the three groups treated with xylazine and the number of spinal segments involved in the area of analgesia when the anaesthetic contained xylazine was significantly greater than with 0.2 mg/kg lidocaine alone ( < 0.01). After the treatment with 0-025 mg/kg xylazine and 0.1 mg/kg lidocaine, flank surgery was performed successfully without additional line block or side effects.

Ultrastructural and immunoelectron microscopic studies of the peri-implant epithelium-implant (Ti-6Al-4V) interface of rat maxilla.

BACKGROUND: The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the peri-implant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface. METHODS: Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy. RESULTS: Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces. CONCLUSIONS: PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

An immunoelectron microscopic localization of noncollagenous bone proteins (osteocalcin and osteopontin) at the bone-titanium interface of rat tibiae.

This study was designed to investigate by postembedding immunogold method the localization and distribution of osteocalcin (Ocl) and osteopontin (Opn) at the bone-titanium interface in rat tibiae 14 and 28 days postimplantation to determine which bone proteins are present at this interface. Both proteins were widely distributed on the newly formed bone and accumulated predominantly in the region of bone close to the titanium, in electron-dense patches in the bone, and at the osteocytic lacunae. Collagenous osteoid showed little or no labeling for either Ocl or Opn. An amorphous zone (20-50 nm) was interposed between the titanium and interfacial slender cells, osteoid, or bone, and was labeled strongly for Ocl but only weakly for Opn. Furthermore, a second electron-dense layer, the lamina limitans, which faces the titanium, was labeled strongly for Opn but weakly for Ocl. Ocl as a marker protein of osteoblasts was sometimes found in the granules and vesicles of the interfacial cells and extracellularly in their intercellular spaces, close to the titanium. However, Opn was not detected in any granules. This is the first report to show that the amorphous zone contains large amounts of Ocl and small amounts of Opn, and that bone contacts titanium through this Ocl-rich amorphous zone. Furthermore, it is suggested that the interfacial cells seem to be osteoblasts, and that Ocl in the amorphous zone is produced and secreted by these cells and functions with Opn as a regulator of the mineralization front close to the titanium, and as a mediator of cell-matrix and matrix-matrix/mineral adhesion along the titanium.

Uncontrolled diabetes hinders bone formation around titanium implants in rat tibiae. A light and fluorescence microscopy, and image processing study.

This study examined the influence of diabetes mellitus on bone formation around cylindrical titanium (Ti) implants (1.0 mm in diameter and 1.5 mm in length) inserted transcortically and extending into the medullary canal of rat tibiae using light and fluorescence microscopies and image processing. Forty-eight male Wistar King A rats (age 5 weeks) were used in this experiment. Streptozotocin was injected intraperitoneally to induce diabetes and the serum glucose concentration was checked to ensure the induction of diabetes prior to implant placement and at the time of sacrifice. The animals were sacrificed 7, 28, 56, or 84 days after placement. Toluidine blue-stained undecalcified sections were prepared for histological observation and image analysis. The Ti implants in the control group became increasingly encapsulated with a bone layer. The implants in the diabetes-induced (DI) group were also surrounded with a thin bone layer. Abundant adipocytes were observed in the DI group as compared with the control group. Quantitative evaluation indicated that the control group showed a significantly higher percent of bone contact, and thickness of surrounding bone and area than the DI group. Consequently, the present study suggests that uncontrolled diabetes would hinder bone formation around Ti implants in rats.

An immunocytochemical study for lysosomal cathepsins B and D related to the intracellular degradation of titanium at the bone-titanium interface.

The morphological relationship between titanium and lysosomal proteinases, cathepsins B and D, at the bone-titanium interface using titanium-coated plastic implants placed for 28 days in the tibiae of 6-week-old rats was immunocytochemically investigated by the colloidal immunogold-silver method. Under light microscopy the titanium layer appeared to make direct contact with the bone and one or a few layers of slender cells were interposed between the bone and titanium. Ultrastructurally, the titanium came in contact with the bone or the slender cell layer through a 20 to 40 nm thin amorphous zone. The slender cells at the bone-titanium interface consisted of two types; one was an osteoblast type with glycogen granules which was found along the newly-formed bone facing titanium layer. The other was a fibroblast type which came in contact with the titanium layer and occasionally endocytosed the detached titanium fragments. In addition, some of the slender cells also showed degenerative changes. Immunocytochemically, cathepsins B and/or D were sometimes colocalized in some phagolysosomes with titanium fragments. These findings suggested that the fibroblast types at the bone-titanium interface may act as scavengers to remove both cell debris and titanium by means of some endocytotic ability, and lysosomal cathepsins also developed in response to the endocytosed titanium. The osteoblast type also appears to show a high degree of osteogenic activity around the titanium-coated plastic implants.

Long-term evaluation of bone-titanium interface in rat tibiae using light microscopy, transmission electron microscopy, and image processing.

We conducted a 2-year histologic and histometric evaluation of the tibial bone-titanium (Ti) implant interface in male rats. Thirty male 6-week-old rats were used in this study. They were divided into two groups: 15 for day 28 and 15 for day 730. Microscopic observation at day 28 revealed that the newly formed bone around the implant almost surrounded the implant, but fibroblastlike cells were interposed in some histologic sections. At day 730, in contrast, such cells were rarely seen, and the bone around the implant presented a lamellar structure. Transmission electron microscopic observation at day 28 disclosed mature or poorly mineralized bone near the implant; however, an electron-dense amorphous zone about 50 nm in thickness was interposed between the bone and Ti. In places slender cells were interposed between the bone and Ti. The amorphous zone was also observed at the cell-Ti interface. At day 730, a poorly mineralized layer remained in some areas between the mature bone and the titanium, and the interposed amorphous zone was still observed. Occasionally, a 200-nm-thick layer, thought to be cell remnant, was seen. As calculated in an image-processing, system analysis, the percent bone contact and the thickness and area of the surrounding bone for the Ti implant at day 28 were 43.6%, 30.4 microns, and 0.10 mm2, respectively, and those at day 730 were 89.9%, 53.5 microns, and 0.19 mm2, respectively. In summary, although the passage of time may affect bone maturity, interfacial cells remain at the bone-Ti interface as a uniform layer together with unmineralized bone.

Histological comparison of early wound healing following dense hydroxyapatite granule grafting and barrier placement in surgically-created bone defects neighboring implants.

The purpose of this study was to examine early wound healing following grafting of dense hydroxyapatite granules (HA granules) and barrier placement in surgically-created bone defects surrounding implants. Eight healthy adult dogs with an average weight of 15 kg were used in this study. Thirty-two bone defects measuring 4 mm x 4 mm were removed with a surgical bur to form continuous bucco-lingual bone defects and 32 implants (16 titanium [Ti]) and 16 hydroxyapatite-coated [HA]) were then placed into the defects. Four implant groups were created: 1) grafting HA; 2) covering with an expanded polytetrafluoroethylene (ePTFE) membrane; 3) grafting HA and covering with ePTFE membrane; and 4) control (no treatment). Animals were sacrificed 28 days after surgery. Histological sections revealed large amounts of newly-formed bone in all bone defects surrounding the implants treated with ePTFE membranes alone. Fibrous encapsulation of HA granules was observed in the defects of the HA granules grafting group. In the group with grafting of HA granules and covering with ePTFE membranes, small amounts of bone tissue were observed among HA granules, but most HA granules were surrounded with fibrous tissue. Bone defects were completely filled with connective tissue in the control group. There were no differences in the histological findings between Ti and HA-coated implants in all cases. Histomorphometric data disclosed that the presence of HA granules in the bone defects significantly arrested bone formation. Our study suggests that the grafting of dense HA into bone defects surrounding implants will result in fibrous healing during the early healing stage.

Immunolocalization of proliferating cell nuclear antigen in the peri-implant epithelium.

The aim of the present study was to investigate the proliferating activity of peri-implant epithelium immunohistochemically using proliferating cell nuclear antigen (PCNA). Eight ITI (Internationale Team für Implantologie) implants were placed into simulated sockets in the mandibles of two beagle dogs two months following tooth extraction. As a control, junctional epithelium of the molar teeth in the same animals were used. The nature of staining and the distribution of PCNA immunoreactivity were determined by scoring a minimum of 100 cells on two sections from each of the implants. In the junctional epithelium, the immunoreactivity to PCNA was detected mainly in the basal cells, in some of the prickle cells, and in a few cells attached to the enamel. In peri-implant epithelium, only some of the basal cells were positive for PCNA. The PCNA score of the peri-implant epithelium was significantly lower than that of junctional epithelium. These results suggest that the peri-implant epithelium maintains a lower capacity to act as a proliferative defence mechanism than does the junctional epithelium.

Failing hollow implants examined by light microscopy and image processing.

The purpose of this study was to evaluate the radiologic, histologic, and histometric findings of three failing hollow implants. On periapical radiographs, these implants showed vertical bone loss up to the hollow portion around the implant. Examination of the histologic sections disclosed that the hollow portions of all the implants were almost filled with bone tissue, although slight bone resorption and presence of granulation tissue infiltrated with inflammatory cells was observed coronal to the hollow portion. Histometric analysis disclosed that the average percent bone contact was 93.1% in case 1, 90.9% in case 2, and 84.3% in case 3 and the average percent bone filling was 42.1%, 50.5%, and 33.8%, respectively. Consequently, there seems to be some potential for successful treatment of these implants because the destructive changes were limited to the coronal aspects of the implant.

Abscess formation around a hydroxyapatite-coated implant placed into the extraction socket with autogenous bone graft. A histological study using light microscopy, image processing, and confocal laser scanning microscopy.

The purpose of this study was to evaluate the radiologic, histologic, and histometric findings for a retrieved hydroxyapatite (HA)-coated implant which had been placed into a fresh extraction socket with autogenous bone graft 3 months previously. A periapical radiography disclosed a vertical bone loss around the implant cervix. Examination of histologic section disclosed that granulation tissue including bone chips around the cervix, and newly-formed bone tissue around the grafted bone tissue on the HA coated surface. In the confocal laser scanning microscopic findings toluidine blue-negative bone tissue showed autofluorescence. Histometric analysis indicated that the average percent bone contact was 29.2% (ranged 26.4% to 34.1%). Suspected reasons for failure were an early exposure of the barrier membrane, its early removal, the implant placement into an infected site, inadequate antibiotic premedication, and/or poor control of infections around teeth prior to implant surgery and around implants before and after placement of barrier membrane.

The effects of diabetes on the interface between hydroxyapatite implants and bone in rat tibia.

We examined the influence of diabetes on the implant-bone interface of hydroxyapatite (HA) implants inserted transcortically and extending into the medullary canal of rat tibiae, and quantitatively assessed the differences in bone reaction using an image processing system. Forty male Wistar King A rats (aged 5 weeks) were used in this experiment; they were sacrificed 84 days after implant placement. Toluidine blue-stained undecalcified sections were prepared for histological observation and image analysis, and the labeled sections were observed by confocal laser scanning microscopy. The HA implants in the bone marrow area in the control group were completely encapsulated with a bone layer, and there were some osteoblast-like cells in the bone lacunae apposing the implant surface. The HA implants in the diabetes-induced (DI) group were partially surrounded with a thin bone layer, and there were some fibroblasts running parallel to the implant surface at areas of no bone contact. Quantitative evaluation indicated that the control group showed significantly higher bone contact rate, bone contact thickness, and bone contact area than the DI group. The DI group showed approximately 30% reduction in the percentage of bone contact and 50% reduction in the thickness and the area of surrounding bone tissue.

Study of bone formation around dense hydroxyapatite implants using light microscopy, image processing and confocal laser scanning microscopy.

The bone reaction to hydroxyapatite (HA) implants inserted transcortically and extending into the medullary canal of rat tibiae was quantitatively assessed using light microscopy, confocal laser scanning microscopy and an image processing system. Sixty-five male rats (6 weeks old) were divided into two groups, 60 for histological observation and image analysis and five for time-labelling. In the histological observation, control sections of 168 days showed a few bone trabeculae in the fatty bone marrow, and Ti implants had become gradually encapsulated with a thick bone tissue layer; however, HA implants became almost completely encapsulated with a thin bone tissue layer during the 168 day experimental period. Histometrical analysis of the percent bone contact revealed that Ti implants showed a continuous increasing curve, and HA implants showed rapid increase in the initial healing period up to 14 days, with 96% bone contact reaching a plateau at 84 days after operation. There was a significant difference in the percent of bone contact between Ti and HA implants throughout the experimental period. Confocal laser scanning microscopic observations revealed the presence of calcein at the 14th day and only slight alizarin colour layer in the bone tissue at the 28th day, both indicating bone formation. These findings suggest that the activity of bone formation was higher at the 14th day than at the 28th day. Also, the percentage of bone contact of HA is superior to titanium throughout the experimental period, and the ascending patterns of both implants are quite different to each other.

Effects of aging on titanium implants inserted into the tibiae of female rats using light microscopy, SEM, and image processing.

We examined the influence of aging on the implant-bone interface of titanium implants inserted transcortically and extending into the medullary canal of rat tibiae, and quantitatively assessed the differences in bone reaction using an image processing system. Three groups of 15 female rats, aged 6 weeks (young group), 22 weeks (adult group), and 80 weeks (old group) were used in this experiment. The animals were sacrificed 28 days after implant placement. Toluidine blue stained undecalcified sections were prepared for histological observation and image analysis, and the implant socket was observed by SEM. There was no difference in the degree of maturation of newly formed bone between the young and adult groups. Titanium implants inserted in the young and adult groups were surrounded with a bone layer. In the old group, however, there was little mature bone tissue around the implants. Quantitative evaluation indicated that the young group showed the highest, the adult group showed a slightly lower, and the old group showed the lowest percent bone contact, thickness of bone contact, and area of bone surrounding the implant.

Histomorphometric analysis of the response of rat tibiae to shape memory alloy (nitinol).

The bone reaction to nitinol (Ni-Ti), a metal with shape memory, and other materials inserted transcortically and extending into the medullary canal of rat tibiae was quantitatively assessed using an image processing system. The materials examined were implants, all of the same shape and size, composed of nitinol, pure titanium (Ti), anodic oxidized Ti (AO-Ti), a titanium alloy (Ti-6Al-4V) and pure nickel (Ni). While the other four implant materials were progressively encapsulated with bone tissues, Ni was encapsulated with connective tissues through the 168-day experimental period, and the Ni implants showed no bone contact at any time during the experimental period. Histometric analysis revealed no significant difference among the tissue reactions to Ti, AO-Ti and Ti-6Al-4V, but Ni-Ti implants showed significantly (P < 0.01) lower percentage bone contact and bone contact area than any of the other titanium or titanium alloy materials.

A study of the regional distribution of bone formed around hydroxyapatite implants in the tibiae of streptozotocin-induced diabetic rats using multiple fluorescent labeling and confocal laser scanning microscopy.

The present study was designed to compare the amount and regional distribution of bone formation around hydroxyapatite (HA) implants in normal (control) rats with that of animals with diabetes mellitus (DM), induced by streptozotocin 2 weeks prior to implant placement. Calcein (CAL), alizarin complexone (AL), and tetracycline (TC) were injected on the 7th, 14th, and 21st days after implantation, respectively, and the rats were sacrificed on the 28th day after implantation. Seventy-microns undecalcified sections of the HA-bone interface in both groups were then prepared for confocal laser scanning microscopy (CLSM) observation. In both groups, bone formation developed from the HA surface to the endosteum, periosteum, or bone marrow. In the control group, around the HA close to the endosteum and periosteum, the new bone showed an extensive lamination pattern of three color layers (CAL, AL, and TC), but in the DM group the labeling density of TC on the 21st day was low. In contrast, on the lateral part of the HA surface (away from the endosteum and periosteum), there was considerably less bone formation in the control group, and in the DM group it was almost completely suppressed. These findings indicate that bone formation around the HA was initiated from the HA surface in the control group, while in the DM group, bone formation along the lateral part of the HA away from the endosteum and periosteum was almost completely suppressed. Furthermore, it is also suggested that in the new bone along the HA close to the endosteum and periosteum, only calcification on the 21st day was depressed.

A histologic evaluation of retrieved hydroxyapatite-coated blade-form implants using scanning electron, light, and confocal laser scanning microscopies.

We histologically examined seven hydroxyapatite-coated (HA) blade implants removed from patients. Four of them radiologically showed severe bone loss and were easily removed with an elevator. Three radiologically showed vertical bone loss and were removed by surgical procedure. Our histological evaluation indicated that coating separation from the HA implants had occurred, and HA coating resorption by bone tissues was suspected in an implant left in situ for 8 years. Several multinucleated giant cells were seen with a few released particles of HA coating at the point lacking bone contact with the HA coating. The presence of microorganisms on and in the HA coating layer was also noted.