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Biomedical subjects

Hideaki Kagami

Publications and source records attributed to Hideaki Kagami.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells.

Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.

Animals↗
PubMed2006

Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering.

Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.

Alkaline Phosphatase↗
PubMed2006

Development of a novel temporary epicardial pacing wire with biodegradable film.

PURPOSE: A temporary epicardial pacing wire (TEPW) has been routinely placed in patients undergoing cardiac surgery. However, its fixation or removal occasionally causes troublesome complications. The aim of this study is to develop a novel TEPW using biodegradable film to fix the electrode to the epicardium without needle stabbing. DESCRIPTION: A biodegradable film was prepared with poly(L-lactide-co-epsilon-caprolactone). The film has a honeycomb-patterned structure that serves as a temporary adhesive for the myocardial surface, and the electrode was incorporated within the film. The novel TEPW was placed on the ventricular epicardium of dogs (group A, n = 5). As a control, conventional TEPW was inserted (group B, n = 6). The pacing threshold, R wave amplitude, impedance, and slew rate were measured at postoperative days 0, 1, 3, 5, 7, and 14, and complications after removal were checked. EVALUATION: All measurements in both groups were identified and differences were not observed. In addition, the novel TEPWs could be easily removed without related complications. CONCLUSIONS: This novel TEPW is safe and feasible for postoperative management of cardiac surgeries.

Absorbable Implants↗
PubMed2006

Preliminary study of tissue-engineered odontogenesis in the canine jaw.

PURPOSE: We previously reported a method for the development of tissue-engineered tooth. However, 1 drawback of the procedure was the inability to determine whether the tooth would function when transplanted in the jaw because it was formed in the omentum of the abdomen. Therefore, the present study was designed to evaluate whether transplantation of dissociated odontogenic cells could induce tissue-engineered odontogenesis in the canine jaw. MATERIALS AND METHODS: Cells were harvested from canine first molar tooth buds and the resulting heterogeneous cell population was seeded on a biodegradable polymer. These constructs were then transplanted into the same sockets after extracting the tooth buds. After transplantation, we evaluated the transplanted constructs using dental x-ray, micro-computed tomography, histology, and immunohistochemistry. RESULTS: After 24 weeks, micro-x-ray computed tomography showed regenerated hard tissues in the jaw, and hematoxylin and eosin staining showed tubular dentin and bone. In the regenerated tissue, osteopontin, osteonectin, and osteocalcin antibodies stained the dentinal matrix. However, enamel tissue and dental-root formation were not observed. CONCLUSION: These data show for the first time the formation of dentin and bone from dissociated odontogenic cells in the canine jaw.

Animals↗
PubMed2006

Constructing a tissue-engineered ureter using a decellularized matrix with cultured uroepithelial cells and bone marrow-derived mononuclear cells.

This study investigated the efficacy of the ureteral decellularized matrix (UDM) as a scaffold material for a tissue-engineered ureter, and the effect of bone marrow-derived mononuclear cells (BM-MNC) on the neovascularization of the scaffold. Canine ureters were treated with deoxycholic acid to remove all cells. Uroepithelial cells (UEC) were obtained from canine bladders, cultured, and then seeded onto the inner surface of the UDM before transplantation into the subcutaneous space of nude mice or the omentum of nude rats. The cultured UECs began showing vacuolar degeneration 3 days after transplantation and gradually disappeared thereafter. To facilitate neovascularization in the implant, BM-MNCs were seeded around the UDM before transplantation. This facilitated the survival of the UECs, which formed three to five cellular layers after 14 days. The mean microvessel density was significantly increased in tissues seeded with BM-MNCs. However, cell-tracking experiments revealed that the increased number of capillaries in the experimental group was not due to the direct differentiation of transplanted endothelial progenitor cells. Our results demonstrate that the UDM is a useful scaffold for a tissue-engineered ureter, especially when seeded with BM-MNCs to enhance angiogenesis.

Animals↗
PubMed2005

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential.

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.

Alkaline Phosphatase↗
PubMed2005

The homeodomain protein PDX1 is required at mid-pancreatic development for the formation of the exocrine pancreas.

The homeoprotein PDX1 is expressed throughout pancreatic development and is thought to play important roles at multiple stages. We describe the properties of a tet-off regulatory scheme to manage the expression of Pdx1 in utero. Cessation of Pdx1 expression at increasingly later gestational times blocked pancreatic development at progressive and morphologically distinct stages and provided the opportunity to assess the requirement for Pdx1 at each stage. Embryonic PDX1 is depleted below effective levels within 1 day of the initiation of doxycycline treatment of pregnant mice. We show that PDX1, which is necessary for early pancreatic development, is also required later for the genesis of acinar tissue, the compartment of the pancreas that produces digestive enzymes. Without PDX1, acini do not form; the precursor epithelium continues to grow and branch, creating a truncated ductal tree comprising immature duct-like cells. The bHLH factor PTF1a, a critical regulator of acinar development, is not expressed and cells producing digestive enzymes are rare. This approach should be generally applicable to study the in vivo functions of other developmental regulators with multiple, temporally distinct roles.

Animals↗
PubMed2005

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↗
PubMed2005

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↗
PubMed2005

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↗
PubMed2004

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↗
PubMed2004

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↗
PubMed2003

High correlation between the bacterial species in denture plaque and pharyngeal microflora.

OBJECTIVES: The morbidity and mortality of the dependent elderly that result from aspiration pneumonia are recognized as a major geriatric health problem. Most cases of bacterial pneumonia are initiated following colonization or superinfection of the pharynx by pathogenic bacteria, followed by aspiration of pharyngeal contents. A recent study revealed that bacteria, that commonly cause respiratory infection, colonized the dentures of dependent elderly. This suggests that denture plaque may function as a reservoir of potential respiratory pathogens to facilitate colonization on the pharynx. The purpose of this study was to determine the possible correlation between denture and pharyngeal microflora. STUDY DESIGN: The denture and pharyngeal bacterial flora of 50 dependent elderly were examined, and the microorganisms identified by culturing. The agreement between the bacterial species in denture plaque and pharyngeal microflora was investigated using the Kappa method. RESULTS: The microorganism species on the dentures and pharyngeal mucosa of the subjects had an agreement rate of 68.5%. The agreement rate for each of the bacterial species of the dentures and pharynx was also demonstrated to be high. CONCLUSIONS: Dentures should be considered an important reservoir of organisations which could colonise the pharynx, and the importance of controlling denture plaque for the prevention of aspiration pneumonia cannot be overemphasized.

Aged↗
PubMed2002

Experimental control of pancreatic development and maintenance.

To investigate the role of the HOX-like homeoprotein PDX1 in the formation and maintenance of the pancreas, we have genetically engineered mice so that the only source of PDX1 is a transgene that can be controlled by the application of tetracycline or its analogue doxycycline. In these mice the coding region for the tetracycline-regulated transactivator (tTA(off)) has replaced the coding region of the endogenous Pdx1 gene to ensure correct temporal and spatial expression of the regulatable transactivator. In the absence of doxycycline, tTA(off) activates the transcription of a bicistronic transgene encoding PDX1 and an enhanced green fluorescent protein reporter, which acts as a visual marker of transgene expression in living cells. Expression of the transgene-encoded PDX1 rescues the Pdx1-null phenotype; the pancreata of these mice develop and function normally. The rescue is conditional; doxycycline-mediated repression of the transgenic Pdx1 throughout gestation recapitulates the Pdx1 null phenotype. Moreover, application of doxycycline at mid-pancreogenesis blocks further development. Adult animals of the rescue genotype that were treated with doxycycline for 3 weeks shut off Pdx1 expression, decreased insulin production, and lost the ability to maintain glucose homeostasis. These results demonstrate the feasibility of controlling the formation of an organ during embryogenesis in utero and the maintenance of the mature organ through the experimental manipulation of a key developmental regulator.

Animals↗
PubMedDate not supplied

Tissue engineering using magnetite nanoparticles and magnetic force: heterotypic layers of cocultured hepatocytes and endothelial cells.

Novel technologies to establish three-dimensional, in vivo-like tissue consisting of various types of cells are required for tissue engineering. We applied magnetic force to construct a heterotypic, layered coculture system of rat hepatocytes and human aortic endothelial cells (HAECs) that was not limited by cell type. Magnetite cationic liposomes carrying a positive surface charge to improve adsorption accumulated in HAECs at a concentration of 38 pg of magnetite per cell. Magnetically labeled HAECs specifically accumulated onto hepatocyte monolayers at sites where a magnet (4000 G) was positioned, and then adhered to form a heterotypic, layered construct with tight and close contact. This cocultured construct significantly (p < 0.05) enhanced albumin secretion by hepatocytes compared with that in homotypic cultures of hepatocytes or heterotypic cocultures of hepatocytes and HAECs without magnets. These results suggest that this novel use of magnetite nanoparticles and magnetic force, which we refer to as "magnetic force-based tissue engineering" (Mag-TE), offers a major advancement in tissue engineering.

Animals↗
PubMedDate not supplied

Construction and harvest of multilayered keratinocyte sheets using magnetite nanoparticles and magnetic force.

Novel technologies to establish three-dimensional constructs are desired for tissue engineering. In the present study, magnetic force was used to construct multilayered keratinocyte sheets and harvest the sheets without enzymatic treatment. Our original magnetite cationic liposomes, which have a positive surface charge in order to improve adsorption, were taken up by human keratinocytes at a concentration of 33 pg of magnetite per cell. The magnetically labeled keratinocytes (2x10(6) cells, which corresponds to 5 times the confluent concentration against the culture area of 24-well plates, in order to produce 5-layered keratinocyte sheets) were seeded into a 24-well ultralow-attachment plate, the surface of which was composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. A magnet (4000 G) was placed under the well, and the keratinocytes formed a five-layered construct in low-calcium medium (calcium concentration, 0.15 mM) after 24 h of culture. Subsequently, when the five-layered keratinocytes were cultured in high-calcium medium (calcium concentration, 1.0 mM), keratinocytes further stratified, resulting in the formation of 10-layered epidermal sheets. When the magnet was removed, the sheets were detached from the bottom of the plates, and the sheets could be harvested with a magnet. These results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach for tissue engineering.

Cell Movement↗