Yoshiya Hashimoto

Process of healing of mucosal defects in the esophagus after endoscopic mucosal resection: histological evaluation in a dog model

BACKGROUND AND STUDY AIMS Resection of a large amount of the esophageal mucosa often causes esophageal ulcer and postoperative stricture. The aim of this study was to evaluate the process of healing of defects in the esophageal mucosa after endoscopic mucosal resection (EMR). MATERIALS AND METHODS Cap-assisted EMR was performed in the thoracic esophagus of six beagle dogs to prepare mucosal defects with a diameter ranging from 15 to 18 mm. The process of mucosal healing was assessed histologically immediately after EMR, and on postoperative day (POD) 2, 4, 7, 14, and 28. RESULTS Immediately after EMR, a thin layer of the submucosa remained in the mucosal defect, and no damage to the muscularis propria was evident. Ulcer formation and inflammatory cell invasion were observed in the remaining submucosa on POD 2 and 4. Angiogenesis and collagen fiber hyperplasia were observed after POD 7. Complete epithelialization of the ulcer was observed on POD 28. In the muscularis propria, further destruction and atrophy were evident after POD 7. Fibrosis of the muscularis propria was observed on POD 28. CONCLUSION In the esophageal wall after epithelial loss resulting from EMR, atrophy and fibrosis of the muscularis propria remain even after epithelialization.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.

Enhanced bone regeneration by gelatin–β-tricalcium phosphate composites enabling controlled release of bFGF

The objective of this study was to investigate the feasibility of biodegradable gelatin–β‐tricalcium phosphate (β‐TCP) composites as a cell scaffold and controlled‐release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and β‐TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical‐sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin–β‐TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and β‐TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF‐free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin–β‐TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF. Copyright

Effects on bone regeneration when collagen model polypeptides are combined with various sizes of alpha-tricalcium phosphate particles

We evaluated the effects on bone formation of combining synthesized collagen model polypeptides consisting of a Pro-Hyp-Gly [poly(PHG)] sequence and alpha-tricalcium phosphate (α-TCP) particles with various median sizes (large: 580.8 μm; small: 136.2 μm; or large and small mixed: 499.3 μm) in a skull defect model in mini-pigs. Quantitative image analyses for the volume density (VD) of new bone revealed that the VD in each α-TCP group was significantly higher than that in the poly(PHG) control group, with the mixed group showing the highest VD among all the groups at 4 weeks after implantation. Histological assessments revealed that the small α-TCP particles were almost completely degraded at 8 weeks. At 12 weeks, all sizes of α-TCP particles were completely degraded and remodeling of the lamellar bone was observed. The present findings suggest that particle size may influence the success of bone formation in defects.

Monomer permeability of disposable dental gloves

STATEMENT OF PROBLEM Studies have suggested that monomers may be able to permeate dental gloves. PURPOSE This study examined the permeability of disposable dental gloves to 6 kinds of dental monomers. MATERIAL AND METHODS The permeability of 6 kinds of dental monomers (methyl methacrylate [MMA], 2-hydroxyethyl methacrylate [HEMA], triethyleneglycol methacrylate [TEGDMA], ethyleneglycol dimethacrylate [EGDMA], urethane dimethacrylate [UDMA], and Bis-glycidyl methacrylate [Bis-GMA]) through 5 kinds of dental gloves (latex, powder-free latex, coated latex, polychloroprene, and polyvinyl chloride) was examined for up to 180 minutes at 37 degrees C. The fingers of unused gloves without pin holes were cut and used in the experiments. Five specimens per test group were examined. One type of monomer was poured into each finger and dipped in ethanol. The ethanol for extraction was measured by a spectrophotometer at a wavelength of 210 nm, and the results were analyzed by analysis of variance and the Kruskal-Wallis test (P<.05). RESULTS Four of the monomers tested (MMA, HEMA, TEGDMA, and EGDMA) permeated the gloves tested, whereas 2 (UDMA and Bis-GMA) did not (P>.01). The amount of monomers permeating the latex in 10 minutes was 0.8 +/- 0.6, 0.6 +/- 0.6, 0.07 +/- 0.1, 0.07 +/- 0.1, 0.1 +/- 0.1 and 0.06 +/- 0.1 microL/mL for MMA, HEMA, EGDMA, TEGDMA, UDMA, and Bis-GMA, respectively. The amount of permeated monomer was then increased in relation to the examination time, and in MMA and HEMA, permeation occurred rapidly during the initial 60 minutes at 3 times the 10-minute values, then continued gradually and linearly. The polyvinyl chloride glove showed the greatest monomer permeability. Two-way analysis of variance showed significant correlations between MMA, HEMA, EGDMA or TEGDMA and UDMA or Bis-GMA (P<.01). Statistical significance was shown between polyvinyl chloride and latex, powder-free latex, coated latex or polychloroprene (P<.01). However, there was no significant relation between any kind of dental monomer and any kind of dental glove. CONCLUSION Within the limitations of this study, 4 of the monomers tested permeated all of the gloves tested.

Evaluation of bone regeneration by porous alpha-tricalcium phosphate/atelocollagen sponge composite in rat calvarial defects

Abstract Purpose Both atelocollagen and alpha-tricalcium phosphate (α-TCP) particles are widely applied as bone graft materials. In this study, we combined atelocollagen and porous α-TCP particles to form an α-TCP/atelocollagen composite sponge (α-TCP/CS), and we evaluated its effects on bone regeneration in rat calvarial defects. Materials and methods α-TCP granules were mixed with a collagen solution. The mixture was poured into plastic molds and then frozen to −80 °C and freeze-dried for 24 h. The composites were then cross-linked in vacuo at 140 °C for 24 h. The obtained composites were characterized by XRD and SEM and observed using micro-CT and histological analysis. Results In vivo micro-CT images at 2, 4, and 6 weeks after surgery showed that in the α-TCP/CS group, the bone volume, bone mineral density, and bone mineral content were higher than those in the groups with only atelocollagen and without scaffolds at 6 weeks after surgery. Histological analysis showed that α-TCP was completely absorbed, and new bone that was continuous along the original bone was observed. Conclusion This study demonstrated that composite sponges created using porous α-TCP particles and atelocollagen were sufficiently adaptable for treating bone defects.

Local Controlled Release of Polyphenol Conjugated with Gelatin Facilitates Bone Formation

Catechins are extensively used in health care treatments. Nevertheless, there is scarce information about the feasibility of local administration with polyphenols for bone regeneration therapy, possibly due to lack of effective delivery systems. Here we demonstrated that the epigallocatechin-3-gallate-conjugated gelatin (EGCG/Gel) prepared by an aqueous chemical synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM) gradually disintegrated with time and facilitated bone formation in a critical size defect of a mouse calvaria. Conjugation of EGCG with the Gel generated cross-linking between the two molecules, thereby leading to a retardation of the degradation of the EGCG/Gel and to a delayed release of EGCG. The prepared EGCG/Gels represented significant osteogenic capability compared with that of the uncross-linked Gel and the cross-linked Gel with uncombined-EGCG. In vitro experiments disclosed that the EGCG/Gel induced osteoblastogenesis of a mouse mesenchymal stem cell line (D1 cells) within 14 days. Using fluorescently-labeled EGCG/Gel, we found that the fraction of EGCG/Gel adsorbed onto the cell membrane of the D1 cells possibly via a Gel-cell interaction. The interaction might confer the long-term effects of EGCG on the cells, resulting in a potent osteogenic capability of the EGCG/Gel in vivo. These results should provide insight into local controlled release of polyphenols for bone therapy.

Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects

PURPOSE Interferon (IFN)-γ is a major cytokine produced by immune cells that plays diverse roles in modulating both the immune system and bone metabolism, but its role in autogenous bone grafting remains unknown. Here, we present that local IFN-γ administration improved the efficacy of autogenous bone graft treatment in an experimental rat model. METHODS An autogenous bone graft model was prepared with critically sized rat calvariae defects. Four weeks (w) after bone graft implantation, rats were treated locally with IFN-γ or were not treated. The effect of IFN-γ on bone formation was evaluated for up to 8w with micro-computed tomography, quantitative histomorphometry, and Von Kossa staining. Osteoclastogenesis was assessed by tartrate-resistant acid phosphatase staining. Immunohistochemistry staining or quantitative polymerase chain reactions were used to estimate the expression of osteoclast differentiation factor and inflammatory cytokines including tumor necrosis factor (TNF)-α, a well-known stimulant of osteoclastogenesis and an inhibitor of osteoblast activity, in defects. RESULTS Newly formed bone gradually replaced the autogenous bone grafts within 4w, although severe bone resorption with osteoclastogenesis and TNF-α expression occurred after 6w in the absence of IFN-γ administration. IFN-γ administration markedly attenuated bone loss, osteoclastogenesis, and TNF-α expression, while it enhanced bone formation at 8w. CONCLUSION Local IFN-γ administration promoted bone formation in autogenous bone grafts possibly via regulating osteoclastogenesis and TNF-α expression. The data provide insights into the potential roles of IFN-γ in autogenous bone grafting.

Porous Alpha-Tricalcium Phosphate with Immobilized Basic Fibroblast Growth Factor Enhances Bone Regeneration in a Canine Mandibular Bone Defect Model

The effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on bone regeneration was evaluated in a canine mandibular bone defect model. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), whereas the other was filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed two, four and eight weeks after implantation. The bone mineral density of the bFGF group was higher than that of the control group at each time point (p < 0.05), and the bone mineral content of the bFGF group was higher than that of the control group at four and eight weeks (p < 0.05). Histological evaluation two weeks after implantation revealed that the porous α-TCP had degraded and bone had formed on the surface of α-TCP particles in the bFGF group. At eight weeks, continuous cortical bone with a Haversian structure covered the top of bone defects in the bFGF group. These findings demonstrate that porous α-TCP with immobilized bFGF can promote bone regeneration.

Effect of an injectable 3D scaffold for osteoblast differentiation depends on bead size

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.