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Biomedical subjects

Minoru Ueda

Publications and source records attributed to Minoru Ueda.

At least 37 records · Page 2Linked to original sources

Ganglioside GD3 promotes cell growth and invasion through p130Cas and paxillin in malignant melanoma cells.

Although ganglioside GD3 levels are highly elevated in malignant melanomas, the role of GD3 in melanomas' malignant properties has not been clearly shown. To investigate this problem, we genetically generated GD3-positive (GD3+) transfectant cells from a GD3-negative (GD3-) mutant line SK-MEL-28-N1 and analyzed the phenotypic changes in the transfected cells. GD3+ cells showed markedly increased cell growth and invasive characteristics. Two bands that underwent stronger tyrosine phosphorylation in GD3+ cell lines than in controls after treatment with FCS were found with molecular masses of 130 and 68 kDa. They were identified as p130Cas and paxillin by sequential immunoprecipitation. Their roles in cell growth and invasion were analyzed with a small interfering RNA (siRNA) approach. Cell growth, as analyzed by BrdUrd uptake, was strongly suppressed in GD3+ cells to near the levels of GD3- cells when treated with siRNA for p130Cas but not when treated with siRNA for paxillin. However, treatment with siRNAs of either p130Cas or paxillin resulted in the marked suppression of the invasive activity of GD3+ cells almost to the levels of control cells. These results suggested that these two molecules function as effectors of GD3-mediated signaling, leading to such malignant properties as rapid cell growth and invasion.

Cell Division↗

Translocation of a 190-kb mitochondrial fragment into rice chromosome 12 followed by the integration of four retrotransposons.

A 190-kb mitochondrial DNA sequence interrupted by seven foreign DNA segments was identified in rice chromosome 12. This fragment is the largest mitochondrial fragment translocated into the rice nuclear genome. The sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome, 45 kb comprising four segments of retrotransposon origin, and 13 kb comprising three segments of unknown origin. The 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence, suggesting that its integration into the nucleus was quite recent. Several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence, suggesting that the past and present arrangements of the mitochondrial genome differ. The four retrotransposons show no mutual sequence similarity and are integrated into different locations, suggesting that their integration events were independent, frequent, and quite recent. A fragment of the mitochondrial genome present in the nuclear genome, such as the 248-kb sequence characterized in this study, is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution.

Biological Transport↗

Osteogenic potential of injectable tissue-engineered bone: a comparison among autogenous bone, bone substitute (Bio-oss), platelet-rich plasma, and tissue-engineered bone with respect to their mechanical properties and histological findings.

Recently, tissue engineering has become available as a regenerative treatment for bone defects. However, the evaluation of its success is limited to histological analysis, and its effects on mechanical hardness remain to be investigated. This study investigated mechanical strength in support of histological findings, specifically for tissue-engineered bone with mesenchymal stem cells (MSCs) and platelet-rich-plasma (PRP). Initially, teeth were extracted, and bone defects on both sides of the mandible were prepared with a trephine bar. The defects were implanted by using the following graft materials: 1) PRP, 2) PRP and dog MSCs (dMSCs), 3) autogenous bone (PCBM), 4) bone substitute (Bio-Oss), and 5) control (defects only). After 2, 4, 8, and 12 weeks of implantation, the defects were histologically assessed to examine their mechanical properties. According to histological observations, the dMSCs/PRP groups had well-formed mature bone compared with the control (defects only), Bio-Oss, and PRP groups. The Vickers hardness test values were 8 (control), 9 (PRP), not detected (Bio-Oss), 11 (PCBM), and 17 (dMSCs/PRP) after 2 weeks. Therefore, tissue-engineered bone can be used for early stage bone regeneration from the viewpoint of histology and mechanical properties.

Animals↗

Effects of ovariectomy and estrogen replacement on rat oral mucosa.

OBJECTIVE: Although a close relationship between the oral discomfort and the menopause is suggested, it is not clear if the alteration of sex steroids would cause any histological or even pathological change in the oral mucosa. The aim of this study was to investigate the possible effect of sex steroid deficiency on the rat oral mucosa as well as the effect of its replacement. METHODS: The oral mucosa of ovariectomized rats was compared to that of sham-operated rats both histologically and immunohistochemically. The same evaluation was also performed after hormone replacement therapy for the ovariectomized rats. RESULTS: The ovariectomized animals presented reduced thickness of the mucosal epithelium with an irregular corneal (keratinised) surface. Partial disappearance of lingual papilla with irregular lete-peg was also noted. The corneal (keratinised) layer in the sublingual region was significantly thinner in ovariectomized animals. The results from immunohistochemistry showed the possibility that the turnover period was prolonged in ovariectomized rats. Estrogen replacement could compensate for the reduction in mucosal thickness in the animal model. CONCLUSIONS: The overall results suggest that sex steroids have a specific role in the maintenance of normal oral mucosa, and its deficiency possibly leads to the onset of oral discomfort in postmenopausal women.

Animals↗

Magnetic force-based mesenchymal stem cell expansion using antibody-conjugated magnetoliposomes.

Recently, there has been an accumulation of evidence indicating that human mesenchymal stem cells (MSCs, multipotent cells resident in the bone marrow) are useful for autologous cell transplantation. However, only small numbers of MSCs have been obtained in bone marrow aspirates. We have developed a novel methodology for enriching and proliferating MSCs from bone marrow aspirates using antibody-conjugated magnetoliposomes (AMLs). The AMLs are liposomes conjugated to anti-CD105 antibody (immunoliposomes) and contain magnetite nanoparticles (diameter 10 nm). In the present study, the AMLs were added to a small volume (1 mL) of human bone marrow aspirate. After a 1-h incubation period, the bone marrow aspirates containing AMLs were seeded into 10-cm tissue culture dishes, and a disk-shaped magnet (diameter 2.2 cm; height 1 cm; 4000 Gauss) was positioned under the dish to enrich MSCs by magnetic force. The MSCs proliferated, forming colonies at the site where the magnet was positioned. In contrast, no colonies and very few viable cells were observed in ordinary culture based on plastic-adherent tendencies of cells without use of AMLs. These results suggest that this AML culture method can rapidly and efficiently expand a small number of MSCs into numbers suitable for clinical application.

Animals↗

Immunohistochemical study of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2, -4 (BMP-2, -4) on lengthened rat femurs.

BACKGROUND: With a hypothesis that "angiogenesis occurs before osteogenesis," an experimental study using a rat model was carried out. Histological and immunohistochemical examinations of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2, -4 (BMP-2, -4) were performed at the margins of bone formation after femoral bone lengthening. MATERIAL AND METHODS: Thirty-five Wistar rats weighing 380-400 g (11-week-old males) were used. An external fixator was applied on the femur, and an osteotomy performed under general anaesthesia. Five days after the operation, femoral lengthening was initiated at a rate of 0.8 mm/day for 8 days. The rats were sacrificed just after distraction was completed, and at 1, 3, 5, 7, 9 and 14 days after distraction. The specimens from these rats were stained with haematoxylin-eosin, VEGF, and BMP-2, -4 immunohistochemical staining, and were investigated. RESULTS: Expression of VEGF was observed in the woven bone at the osteogenetic front and near to osteoblasts around the newly formed bone. On the other hand, expressions of BMP-2, -4 were seen in the hypertrophic chondrocytes. In the same specimen, the VEGF area was further away from the bone stump than the BMP-2, -4 areas. CONCLUSION: These results confirm the hypothesis that angiogenesis is induced before osteogenesis.

Animals↗

Heat and mechanical hyperalgesia in mice model of cancer pain.

We developed a mouse model of cancer pain to investigate its underlying mechanisms. SCC-7, squamous cell carcinoma (SCC) derived from C3H mice, was inoculated subcutaneously into either the plantar region or thigh in male C3H/Hej mice. Heat and mechanical sensitivity as well as spontaneous behavior were measured at the plantar surface of the ipsilateral hind paw after the inoculation. Inoculated sites were histologically examined, and the expression of capsaicin receptors (TRPV1) was examined in the dorsal root ganglia (DRG) to clarify their potential contribution to pain sensitivity. Inoculation of cancer cells induced marked heat hyperalgesia and mechanical allodynia in the ipsilateral hind paw for two weeks in both plantar- and thigh-inoculation models. Signs of spontaneous pain, such as lifting, licking and flinching of the paw were also observed. However, further growth of the tumor reversed the mechanical allodynia in both plantar- and thigh-inoculation models, and heat hyperalgesia in thigh-inoculation models. Histologically, no infiltration of the tumor cells into the nerve was observed. TRPV1 immunoreactive cells increased in the L5 DRG on day 7, but returned to the control level on day 15 post-inoculation. Intraperitoneal administration of the competitive TRPV1 antagonist capsazepine inhibited hyperalgesia induced by tumor cell-inoculation in either plantar- or thigh-inoculated animals. This study indicated that inoculation of SCC resulted in spontaneous pain, heat hyperalgesia and mechanical allodynia. The altered expression of TRPV1 in the DRG may be involved in behavioral changes in this model.

Animals↗

Effects of cryopreservation on histology and viability of cultured corneal epithelial cell sheets in rabbit.

PURPOSE: To increase the availability of cultured corneal epithelial cell sheets as a replacement for corneal limbal allograft, the effects of cryopreservation on viability of cultured corneal epithelial cells were investigated. METHODS: Normal rabbit corneal limbal tissue was excised, and the cells were cultured with mitomycin C-treated 3T3-J2 cells as a feeder layer. Stratified corneal epithelial cell sheets were obtained within 20 days. All sheets with a diameter of 8 mm were punched out with a biopsy punch and stored in either 10% glycerol or dimethyl surfoxide (DMSO) at -80 degrees C or -196 degrees C for 4 or 12 weeks. After thawing, the sheets were evaluated histologically, and cell viability was analyzed by colorimetric cell viability assay using a tetrazolium salt. RESULTS: Structural damage such as vacuolar degeneration was more clearly observed in the corneal epithelial cell sheets cryopreserved with DMSO than those with glycerol, especially at -80 degrees C, whereas only minor morphologic changes were observed in the corneal epithelial cell sheets cryopreserved in glycerol at both temperatures. Colorimetric cell viability assay revealed that the storage conditions at the lower temperature (-196 degrees C) showed higher cell survival than those at the higher storage temperature (-80 degrees C). The difference between the two cryoprotectants, however, was not significant. Among the conditions used in this study, the samples cryopreserved with glycerol at -196 degrees C showed the highest cell survival rate (70.3 +/- 8.3% and 66.4 +/- 14.7% for 4 and 12 weeks, respectively). The difference between this group and those stored at -80 degrees C was significant for both 4 weeks and 12 weeks of storage using either glycerol or DMSO. Although the cryopreserved cell sheets could not maintain their original layered structure after thawing, viable cell sheets could be regenerated. CONCLUSIONS: The cell survival rates obtained after freezing storage were reasonable; however, the cryopreserved sheets could not maintain their original layered structure. More work needs to be done to better preserve the corneal epithelial cell sheets.

Animals↗

Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits.

In the present study, we compared bone regeneration ability in sinus floor elevation between a tissue engineering method using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP), and a promising new method using particulate cancellous bone and marrow (PCBM) and PRP. Bilateral sinus floor elevation procedures were performed in 18 adult Japanese white rabbits. MSCs/PRP or PCBM/PRP complexes were grafted to each maxillary sinus in the same rabbits. The MSCs were isolated from rabbit iliac crest marrow, and PRP was obtained from peripheral blood. PCBM were collected from the rabbit iliac crest and mixed with PRP. The animals were sacrificed at 2, 4, and 8 weeks after transplantation, and the bone formation ability of each implant was evaluated histologically and histometrically. According to the histological observations, both sites (MSCs/PRP and PCBM/PRP) showed well newly formed bone and neovascularization at 2 and 4 weeks. However, at 8 weeks, the lamellar bone was observed to be occupied by fatty marrow in large areas in both sites. There was no significant difference in bone volume or augmented height between MSCs/PRP and PCBM/PRP groups each week, but there were significant differences in bone volume and augmented height between 2 and 8 weeks in PCBM/PRP or MSCs/PRP groups and in bone volume between 4 and 8 weeks in the PCBM/PRP group (P<0.05). These results suggest that the MSCs/PRP complex may well be used for bone regeneration in sinus floor elevation, compared with the PCBM/PRP complex.

Animals↗

Histological and immunohistochemical studies of tissue engineered odontogenesis.

The successful regeneration of complex tooth structures based on tissue-engineering principles was recently reported. The process of this regeneration, however, remains poorly characterized. In this study, we have used histochemistry to examine the regeneration process of tissue engineered teeth in order to determine the cell types that give rise to these engineered tooth structures. Porcine third molar tooth buds were dissociated into single-cell suspensions and seeded onto a biodegradable polyglycolic acid polymer scaffold. Following varying periods of growth in rat hosts, the specimens were evaluated by histology and immunohistochemistry. Aggregates of epithelial cells were first observed 4-6 weeks after implantation. These aggregates assumed three different shapes: a natural tooth germ-like shape, a circular shape, or a bilayer-bundle. Based on the structure of the stellate reticulum in the dental epithelium, the circular and bilayer-bundle aggregates could be clearly classified into two types: one with extensively developed stellate reticulum, and the other with negligible stellate reticulum. The epithelial cells in the circular aggregates differentiated into ameloblasts. The continuous bilayer bundles eventually formed the epithelial sheath, and dentin tissue was evident at the apex of these bundles. Finally, enamel-covered dentin and cementum-covered dentin formed, a process most likely mediated by epithelial-mesenchymal interaction. These results suggest that the development of these engineered teeth closely parallels that of natural odontogenesis derived from the immature epithelial and mesenchymal cells.

Ameloblasts↗

GM1 expression in H-ras-transformed NIH3T3 results in the suppression of cell proliferation inducing the partial transfer of activated H-ras from non-raft to raft fraction.

c-H-ras is located in lipid/rafts and undergoes cholesterol dependent regulation. To analyze the regulatory effects of ganglioside GM1 on the proliferation of fibroblasts transformed with mutated ras gene, GM1 synthase cDNA was transfected into NIH3T3/H-ras cells containing mutation. In the transient expression system with EGFP-fused GM1 synthase, the ratio of BrdU-positive cells among EGFP-positive cells was compared between GM1(+) transfectant cells and control cells, indicating that the transient GM1 expression suppresses cell proliferation. Then, established transfectant cells C21 and C34 expressed definite levels of GM1, and analyzed for the cell growth with the control cells D2 and D4 expressing no GM1. GM1(+) cells showed reduced proliferation compared with controls. Phosphorylation levels of ERK1/2 after FCS treatment were examined, showing that those on the GM1(+) transfectant cells increased slowly, while those in the controls rapidly reached the plateau. Fractionation of Triton X-100 extracts with sucrose density gradient ultra-centrifugation revealed that activated H-ras proteins from controls as well as NIH3T3/H-ras were completely localized in non-GEM/raft fraction. On the other hand, some portions of activated H-ras were transferred to GEM/raft fraction, i.e., 32% in C21, and 8% in C34. Since the Ras effector Raf-1 was localized in non-GEM/raft, the growth suppression might be caused, at least partly, by the movement of activated H-ras to GEM/raft fraction.

Animals↗

Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneous implant placement.

This clinical study was undertaken to evaluate the use of tissue-engineered bone, mesenchymal stem cells, platelet-rich plasma, and beta-tricalcium phosphate as grafting materials for maxillary sinus floor augmentation or onlay plasty with simultaneous implant placement in six patients with 3- to 5-mm alveolar crestal bone height. All 20 implants were clinically stable at second-stage surgery and 12 months postloading. A mean increase in mineralized tissue height of 7.3+/-4.6 mm was evident when comparing the pre- and postsurgical radiographs. Injectable tissue-engineered bone provided stable and predictable results in terms of implant success.

Absorbable Implants↗

Immunohistochemical localization of cytokeratin 19, involucrin and proliferating cell nuclear antigen (PCNA) in cultured human gingival epithelial sheets.

It has been suggested that human cultured gingival epithelial sheets may serve as a possible grafting material. The purpose of this study was to examine the biological characteristics of human cultured gingival epithelial sheets by epithelial differentiation and proliferation markers. Immunohistochemical localization of cytokeratin 19, involucrin and proliferating cell nuclear antigen (PCNA) were examined in human cultured gingival epithelial sheets samples from twenty patients. Cytokeratin 19-immunopositive cells were scattered mainly in the suprabasal layer. Immunoreactivity for involucrin was observed in all layers except for the basal layer. The majority of proliferating cell nuclear antigen-immunopositive cells was found in the basal layer. These results suggested that the cultured human gingival epithelial sheets were biologically active and in proliferative condition, which implies that this biological product may be a potential grafting material.

Adult↗

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

STUDY DESIGN: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. OBJECTIVES: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. SUMMARY OF BACKGROUND DATA: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. METHODS: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. RESULTS: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. CONCLUSION: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Animals↗

The effect of the long-term cultivation on telomere length and morphology of cultured epidermis.

BACKGROUND: Cultured epidermis has been successfully used in clinical treatment such as burns and pigmentary disorders. Although the generation of wide cultured epidermis for clinical use may require repeated passages, especially for allografts, the effects of long-term cultivation on its quality and cell viability are not well known. OBJECTIVES: To investigate the changes in morphology, telomere length, and telomerase activity during the passages of cultured epidermis and keratinocytes up to the passage limit, and to examine the usefulness of telomere length as a performance criterion for cultured epidermis. METHODS: The keratinocytes obtained from five patients were used to generate cultured epidermis. At the early passage and after cultivation up to the passage limit, morphology, telomere length and telomerase activity were investigated by using microscopes, southern blot analysis and telomeric repeat amplification protocol assay, respectively. RESULTS: The cultured cells started to show morphological changes when each passage was close to its limit and the cell sheets assumed an irregular stratification with various sizes of cytoplasm and nuclei. At the passage limit, the telomere length had decreased approximately 80-85%, and the average telomerase activity had declined under serum-free culture conditions. CONCLUSION: The results of this study showed the morphological change and telomere length reduction by long-term cultivation on cultured epidermis. Although the reduction in telomere length and telomerase activity may not be the major cause of the senescence, they could provide a useful information for the quality of the cultured epidermis.

Cells, Cultured↗

Accelerating effects of basic fibroblast growth factor on wound healing of rat palatal mucosa.

PURPOSE: In this study, basic fibroblast growth factor (bFGF) was examined for its ability to accelerate tissue repair in a rat oral mucosal wound. MATERIALS AND METHODS: A 4-mm mucosal defect was surgically made to the depth of the periosteum in a rat palate. bFGF was injected along the edge of the mucosal defect immediately after surgery. A control group received only phosphate-buffered saline vehicle. RESULTS: bFGF significantly accelerated granular tissue formation and reepithelialization. From the histologic analysis, the bFGF-treated group showed relatively faster collagen maturation. Starting 3 days after surgery, fibroblast growth factor receptor 1 (FGFR1)-positive cells appeared in the granular and spinous cell layers of the reepithelializing mucosa in the bFGF-treated group, whereas almost none was observed in the intact oral mucosa. By day 5, FGFR1-positive cells were seen below the stratum corneum, even in the control group. However, the number and intensity of FGFR1-positive cells in the bFGF-treated group were greater than in the control group. Results of immunostaining against proliferating cell nuclear antigen showed that bFGF stimulated cell proliferation of the basal cell layer in the regenerating epithelium. At a higher dose of bFGF, proliferating cell nuclear antigen-positive cells were also observed in the submucosal connective tissue. CONCLUSION: By the induction of its ligand protein concomitant with direct effects such as increased granular tissue formation and reepithelialization, a single topical application of bFGF facilitated wound healing in rat oral mucosa. The results of this study support the consideration for bFGF application for patients with impaired healing of oral mucosal injury.

Analysis of Variance↗

Cartilage formation by serial passaged cultured chondrocytes in a new scaffold: hybrid 75:25 poly(L-lactide-epsilon-caprolactone) sponge.

PURPOSE: This study was designed to determine whether multipled chondrocytes immersed in a new scaffold, 75:25 poly(L-lactide-epsilon-caprolactone) sponge coated with type I collagen (75-PLC scaffold), could be used to generate cartilage tissue in vivo and to evaluate the correlation between cartilage generation and the phenotype of the proliferated chondrocytes. MATERIALS AND METHODS: Rat chondrocytes were suspended in 75-PLC scaffold at a density of 1 x 10 7 cells/mL after proliferation in a monolayer for 1 (P1) to 4 passages (P4) and implanted in nude mice for 4 weeks. Cells were characterized by the expression of genes encoding type II collagen, aggrecan, and type I collagen by Northern hybridization, and consequently, the newly formed tissue was evaluated histologically. RESULTS: The expression of aggrecan messenger RNA gradually decreased with the passaged cultures; however, the expression of type I collagen messenger RNA increased with time. The cartilage formations in all specimens were found not only in P1 chondrocytes but also in P2 chondrocytes, although when P3 chondrocytes were grafted, approximately 50% of cartilage formation was still observed up to but not beyond P4. CONCLUSION: It is suggested that cartilage tissue is generated with cultured chondrocytes up to P2 but not beyond P4. Northern blot analysis is useful for the assessment of whether the cells are capable of regeneration.

Absorbable Implants↗

Horizontal alveolar distraction of the narrow maxillary ridge for implant placement.

PURPOSE: The purpose of this report was to describe a surgical technique for performing horizontal alveolar distraction of the knife-edge maxillary ridge. PATIENT AND METHODS: The patient was a 17-year-old woman with atrophy of the alveolar rim in the anterior upper jaw, which had inadequate width for implant placement. The transport segment was constructed by the osteotomy of the labial cortex of the alveolus. A transport plate of a distractor (LEAD system; Stryker Leibinger, Kalamazoo, MI) was placed on the transport segment. The distraction rod was inserted horizontally, and put in contact with the palatal cortex at the top. A base plate was not placed. RESULTS: The alveolar distraction was successfully performed to gain 6.0 mm in width and 0.5 mm in height, allowing placement of three 14-mm implants. All the implants were integrated so as to support the prosthesis. CONCLUSIONS: Alveolar distraction can be useful for augmenting the narrow ridge horizontally and placement of implants.

Adolescent↗