Biomedical subjects

Shohei Kasugai

Publications and source records attributed to Shohei Kasugai.

At least 19 recordsLinked to original sources

PubMed2007

Histological and histomorphometrical comparative study of the degradation and osteoconductive characteristics of alpha- and beta-tricalcium phosphate in block grafts.

The purpose of the present study was to compare alpha- and beta-tricalcium phosphate (TCP) as bone graft material for augmenting highly resorbed alveolar ridges. The cranial bones of 15 rabbits were used. Three titanium chambers filled with porous blocks of alpha-TCP, beta-TCP, or blood clots were placed in each slit. The two TCP blocks had similar inner/outer structures and purities. Animals were sacrificed after 2, 4, and 8 weeks. Specimens were embedded in polyester resin as nondecalcified specimens, and evaluated both histologically and histomorphometrically. In both TCP groups, blocks had hardly degraded at 2 weeks while in the alpha-TCP group, the block had notably started degrading after 4 weeks. In the beta-TCP group, degradation began at 4 weeks and this degradation had increased just slightly after 8 weeks. The alpha-TCP block degraded significantly more than the beta-TCP block. Residual alpha-TCP particles surrounded by newly formed bone decreased over time, and both particles and newly formed bone were simultaneously absorbed by osteoclast-like cells. These observations suggest that residual alpha-TCP particles surrounded by newly formed bone may disappear progressively from bone and could be incorporated into the bone remodeling cycle in combination with newly formed bone.

Absorbable Implants↗

PubMed2006

Biodegradation process of alpha-TCP particles and new bone formation in a rabbit cranial defect model.

The purpose of the present study was to observe the biodegradation process of pure alpha-tricalcium phosphate (alpha-TCP) particles and to determine the efficacy of alpha-TCP as a space maintainer in a bone defect. We used 14 rabbits and prepared two cranial bone defects in each rabbit. One defect was left empty as a control, whereas the other was filled with alpha-TCP particles about 300 mum in diameter. Animals were sacrificed at 1 week, 4 weeks, and 8 weeks. The cranial bone was then embedded either in paraffin wax for the preparation of decalcified specimens, or in polyester resin for the preparation of nondecalcified specimens. All specimens were evaluated histologically and histomorphometrically. As a consequence of the degradation of alpha-TCP, a "reticulate structure" appeared in the particles at 1 week and new bone was observed in this structure at 8 weeks. The amount of new bone between the control and experimental groups was not significantly different at any of the time points. However, in the experimental group, new bone at the surface of alpha-TCP was evident even in the center of the defect whereas fibrous connective tissue was dominant in the control group. These results indicate that alpha-TCP is a degradable osteoconductive material that is able to act as a space maintainer for bone regeneration when applied to a bone defect. While there was no significant difference in total bone formation between the experimental and negative control groups, the space-maintaining and osteoconductive properties of the particles may result in more complete bone formation in longer-term studies.

Absorbable Implants↗

PubMed2006

Bone regeneration by modified gene-activated matrix: effectiveness in segmental tibial defects in rats.

Gene-activated matrix (GAM) is a matrix, such as collagen-containing plasmid vector, that encodes a protein to stimulate tissue regeneration. In the original GAM system, gene transfer efficiency was extremely low. We have recently reported that modifying GAM with calcium-phosphate precipitates (CaP) enhances the efficiency of gene transfer. The purpose of this study was to evaluate the effects of our modified GAM on tissue regeneration. We prepared critical size segmental bone defects in rat tibiae and transplanted GAM consisting of bovine atelocollagen and expression plasmid vector (bmp2), which encodes human BMP2, with or without CaP. The tibiae were later examined radiographically, histologically, and mechanically. Implantation of bmp2-CaP-collagen at 12 microg bmp2 bridged the bone defect at 4 weeks, and the strength of the bone was comparable to that of an intact tibia at 6 weeks. Implantation of bmp2-collagen at the same dose of bmp2 bridged the defect to a smaller extent. Neither collagen alone nor vacant vector-CaP-collagen bridged the defect. These results indicate that our modified GAM with CaP has the potential to be effective in tissue regeneration at lower plasmid DNA doses than used in previous studies.

Animals↗

PubMed2006

Platelet separation from whole blood in an aqueous two-phase system with water-soluble polymers.

Platelet-rich plasma (PRP) stimulates tissue healing and centrifugation is the only method for PRP preparation. The purpose of the present study was to develop a method to separate platelets without centrifugation. We used 16 polymers of different chemical characteristics and mixed each polymer with fresh whole blood containing anticoagulant citrate-dextrose. Then, we observed blood cell separation. Focusing on the effective polymers and comparing our method with the conventional centrifugation method, we examined platelet recovery rate and P-selectin expression, which represents platelet activation. Poly-L-glutamic acid (PGA) and poly-L-aspartic acid (PAA) separated platelets efficiently; however, these polymers activated platelets. On the contrary, poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB), which has phosphorylcholine residues mimicking a cellular membrane molecule, separated platelets moderately and the PMB-separated platelets were nearly inactive compared to the separation with the ordinary centrifugation and PGA. Conclusively, the present experiments demonstrated that without centrifugation platelets can be separated from whole blood with some water-soluble polymers, such as PGA, PAA, and PMB, and that PMB has an advantage not to activate platelets.

Biopolymers↗

PubMed2006

[Dental implant treatment to osteoporosis patients].

Prosthetic treatment with dental implant is currently reliable. Since dental implant treatment can yield much satisfaction to patients compared to other treatments, such as bridge or removable denture, number of patients, who desire to be treated with dental implant, is increasing. Cortical bone of female mandible is thin compared to that of male mandible and postmenopausal condition decreases bone density of the cancerous bone. Furthermore, estrogen-deficiency delays implant and bone connection. Thus, it is necessary to treat osteoporotic patients with dental implants carefully compared to usual patients.

Dental Implantation, Endosseous↗

PubMed2006

Effects of exposure to 4-META/MMA-TBB resin on pulp cell viability.

Adhesive restorative systems have expanded the range of possibilities for direct pulp-capping technique, with evidences of clinical success in vital pulp therapy. However, quite few studies have described the direct responses of pulp cells following the application of resinous materials to pulp exposure. To address this issue, effects of exposure to an adhesive resin, 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin on cellular activity were investigated in an established rat dental pulp cell line (RPC-C2A). RPC-C2A cells were cultured on normal plastic plates or the disks prepared from 4-META/MMA-TBB resin (Super Bond C&B) in a-MEM containing 10% FBS. After 3, 7 and 14 days, DNA content and alkaline phosphatase (ALP) activity were measured. Total RNA in each group was extracted and RT-PCR analysis was performed. Moreover, the live cell ratio was also evaluated by cytotoxicity assay after treatment with various concentrations of 4-META/MMA-TBB. At day 3, 7 and 14, amount of DNA and ALP activity of the cells on normal plastic plates and the one on the 4-META/MMA-TBB were comparable. Cells of both groups expressed mRNA of type I collagen (Coll), ALP, osteopontin (OPN), osteocalcin (OC), and bone morphogenetic protein (BMP-2). Furthermore, 4-META/MMA-TBB (10(-1)% or less) did not influence dead cell ratio in the confluent state. According to the results of these in vitro studies, exposure to this resinous material would not induce cytotoxic response in the pulp cells.

Alkaline Phosphatase↗

PubMed2005

Thermotolerance of pulp cells and phagocytosis of apoptotic pulp cells by surviving pulp cells following heat stress.

Apoptosis is known to be associated with wound healing and regeneration of dental pulp. We examined the effects of heat stress on clonal dental pulp cell line (RPC-C2A cells) to clarify the pulp wound healing process. RPC-C2A cells were exposed to heat stress at 43 degrees C for 45 min. After several time intervals, the inhibition of cell proliferation and apoptosis induction were analyzed by cell viability assay, DNA gel electrophoresis, nuclear staining, and terminal deoxynucleotidyl transferase mediated labeling assay. RPC-C2A cells showed the thermotolerance following heat stress. We found that apoptosis was induced in some RPC-C2A cells, whereas others remained alive, and observed the engulfment of apoptotic cells by scavenger-like RPC-C2A cells following heat stress. We also analyzed the phagocytotic activity of RPC-C2A cells and found that they had an ability to engulf apoptotic RPC-C2A cells, which was stimulated by heat stress. These results suggest that heat stress induces apoptosis of RPC-C2A cells, which are phagocytosed by the surviving RPC-C2A cells.

Animals↗

PubMed2005

A new technique with calcium phosphate precipitate enhances efficiency of in vivo plasmid DNA gene transfer.

In vivo gene transfer with plasmid vector has been applied experimentally and clinically; however, the low level of gene transfer efficiency with plasmid vector is a problem. We speculated that the combination of calcium phosphate precipitate (CaP) and plasmid vector could solve this problem because CaP stabilizes plasmid DNA. In the present study, we used a plasmid exression vector encoding enhanced green fluorescent protein and combined the vector with CaP. Then, this combination was mixed with bovine type I atelocollagen. After incubating this mixture in phosphate-buffered saline, the amount of the plasmid DNA in the supernatant was low when the plasmid DNA was combined with CaP. Furthermore, the plasmid DNA, which was combined with CaP, was stable in DNase digestion in vitro. The plasmid vector with or without CaP, together with the atelocollagen, was transplanted subcutaneously or injected in the bone marrow of the femurs of rats. Then, the fluorescence was observed under a confocal laser scanning microscope and the fluorescence intensity in the tissue homogenates was measured. In these animal experiments, the fluorescence was extensive when the plasmid DNA was combined with CaP. These results indicate that our formula, collagen/CaP/DNA, appeared efficient for in vivo gene transfer.

Animals↗

PubMed2005

Molecular and cell biological properties of mouse osteogenic mesenchymal progenitor cells, Kusa.

A cell line of murine osteogenic progenitor cells, Kusa, was established from femoral bone marrow stromal cells with other types of mesenchymal progenitor cells. We characterized two sublines of Kusa (Kusa-A1 and Kusa-O) from several aspects, including the use of an expression profiling system, a cDNA microarray. The original Kusa subline (Kusa-A1) had high alkaline phosphatase activity and high accumulation of calcium deposits in a condition inducing mineralization, with ascorbic acid and beta-glycerophosphate. Kusa-O, a low osteogenic subline of Kusa, had high alkaline phosphatase activity but slow accumulation of calcium deposits even in the inducing condition. These two Kusa sublines differed in the expression of the osteogenic marker genes, osteocalcin and osteopontin, during mineralization. A type of cDNA microarray revealed marked downregulation of gene expression in the inducing condition in both Kusa-A1 and Kusa-O. Another type of high-throughput microarray was performed to examine the difference in gene expression patterns between Kusa-A1 and Kusa-O. By this analysis, periostin, which would be involved in a stage of osteogenesis, was low in Kusa-A1. On the contrary, Myocyte enhancer factor 2C (MEF2C), a myogenic transcriptional factor, was high in Kusa-A1, although no expression of any other myogenic genes was shown.

Alkaline Phosphatase↗

PubMed2005

[Bone augmentation at present and in the near future].

Autologous bone graft is a gold standard for bone augmentation and is routinely applied. As methods for bone augmentation in the dental field, GBR, distraction osteogenesis, bone substitutes of calcium-phosphate materials and PRP have already been applied clinically. Furthermore, clinical results of the applications of recombinant proteins of BMP and various growth factors, and the applications of the cells from bone marrow and periostium have been reported. A new method for bone augmentation is thus likely to appear. Society accepts medical treatments that are "effective", "safe", "simple" and "economical". Taking the initials of these words, this paper proposes the concept of "2E2S". Based on recent studies of bone augmentation from the viewpoint of "2E2S", the author will predict bone augmentation in the near future.

Bone Regeneration↗

PubMed2005

[Local application of statin for bone augmentation].

It has been reported that statin, an inhibitor of cholesterol synthesis, enhances bone formation by stimulating osteoblastic differentiation and function and that it also inhibits bone resorption by osteoclasts. Although the potentiality of statin as a therapeutic drug for osteoporosis has been expected, the clinical evidences supporting this potentiality are few. On the other hand, there are several reports demonstrating that local application of statin promotes bone augmentation and bone regeneration. The history of statin as a therapeutic drug for hypercholesterolemia is long and the toxicity of this drug has been well examined. Thus, it is unlikely that local application of this drug at low dose will induce serious side effects. It is possible that local bone augmentation with statin will appear clinically in the near future if proper local drug delivery system for statin is developed.

Animals↗

PubMed2005

Evaluation of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated dressing on surgical wounds.

The ideal dressing material is bio-inert and keeps the wound site moist. It is equally important that no regenerative tissue is peeled off on the removal of the dressing. 2-Methacryloyloxyethyl phosphorylcholine (MPC) has a phospholipid polar group that mimics a biomembrane. We prepared poly [MPC-co-n-dodecyl methacrylate (DMA)] (PMD), using conventional radical polymerization with 2,2'-azobisisobutyronitrile as an initiator, and coated it on polyurethane (PU; Tecoflex 60 Thermedics Inc.) membrane. Full-thickness surgical wounds were made on the dorsal skin of rats and wound healing was compared under the following three conditions: air-exposed control (no dressing), PU dressing, and PMD dressing. At 3, 4 and 7 days after the operation, the wound sizes of the PMD dressings were smaller than the non-dressed wound, and at 6 and 7 days after the operation, the wound sizes of PU dressing were smaller than that of the air-exposed group. But there were no significant difference between the PMD dressing group and PU dressing group. Histologically, scab formation was not observed on the PU or PMD-dressed wounds. However, in the air-exposed control, a scab was formed and re-epithelialization of the wound site was prevented. Additionally, no damage was observed in the histological section of PMD dressed wound after the wound was cured. These results indicate that PMD dressing (PMD-coated PU membrane) has the potential to provide an inert environment for wound healing as well as PU.

Animals↗

PubMed2004

A simple murine model for immobilization osteopenia.

Reduction of loading force to bone induces osteopenia. Although the tail suspension model is the frequently used osteopenia model, this model burdens the animals with nonphysiologic blood distribution and systemic stress. We developed a new simple animal model for osteopenia under reduced loading. Both hind legs of male ICR mice (8 weeks old) in the experimental group were inserted into plastic tubes, which then were connected with wires of three sizes. This apparatus completely immobilized both femurs while allowing the tibias to move. Animals were pair-fed and sacrificed on Days 3, 7, 10, and 14 after immobilization. Bone mineral density measurement with dual energy xray absorptiometry revealed bone loss in the immobilized femurs. Histomorphometric analysis showed increased bone resorption and decreased bone formation, which started from Day 7 and continued until Day 14, resulting in structural disorders in the cancellous bone. Osteoclast population increase before osteoblast population decrease revealed that osteoclasts initially affect the process of this type of osteopenia. Our immobilization model is simple, easy to use, well-tolerated by the animal, and has potential for evaluating the therapeutic effects of drugs to treat osteopenia caused by reduced loading.

Animals↗

PubMed2003

Inhibition of osteoclastogenesis by a phosphodiesterase 4 inhibitor XT-611 through synergistic action with endogenous prostaglandin E2.

We examined the effect of a phosphodiesterase 4 (PDE4) inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611) on osteoclast formation in three different mouse bone-marrow cell (BMC) culture systems. We confirmed that selective inhibitors of PDE4, including XT-611, among several PDE inhibitors decreased osteoclast formation in the BMC culture system. XT-611 also inhibited osteoclast formation in co-culture of mouse bone-marrow stromal cell line ST2 and adherent cell-depleted (ACD)-BMCs. However, it did not inhibit osteoclastogenesis in culture of ACD-BMCs alone in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-kappaB ligand (sRANKL). XT-611 significantly increased prostaglandin E(2) (PGE(2)) production from ST2 cells and, in combination with PGE(2), synergistically increased cAMP concentration in osteoclast progenitors. In the ST2 co-culture system, XT-611 did not influence the expression of RANKL, osteoprotegerin and RANK mRNAs. By combined treatment with XT-611 and PGE(2) of ACD-BMCs, osteoclast multinucleation was clearly inhibited with decrease in the expression of calcitonin receptor mRNA, while the expression of RANK and c-fms (an M-CSF receptor) mRNAs was unchanged. These results indicate that the PDE4 inhibitor inhibits osteoclastogenesis by acting on osteoclast progenitors synergistically with PGE(2) secreted from stromal cells, but not by influencing the cell-to-cell interaction between stromal cells and osteoclast progenitors.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

The effects of enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect.

OBJECTIVE: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. MATERIALS AND METHODS: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2'-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. RESULTS: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. CONCLUSION: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.

Alkaline Phosphatase↗

PubMed2003

S100A4: a novel negative regulator of mineralization and osteoblast differentiation.

S100A4 is an intracellular calcium-binding protein expressed by osteoblastic cells. However, its roles in bone physiology are unknown. Because before matrix mineralization, its expression is markedly diminished, we hypothesized that S100A4 negatively regulates the mineralization process. In this study, we investigated the effects of the inhibition of S100A4 synthesis on osteoblast differentiation and in vitro mineralized nodule formation. Inhibition of S100A4 synthesis was achieved by an antisense approach in the mouse osteoblastic cell line MC3T3-E1. Cell clones that synthesized low levels of S100A4 (AS clones) produced markedly increased number of mineralized nodules at much earlier stages in comparison with controls as demonstrated by Alizarin red S and von Kossa staining. The expression of type I collagen (COLI) and osteopontin (OPN) increased in AS clones compared with controls. Bone sialoprotein (BSP) and osteocalcin (OCN), molecules associated with mineralization and markers for mature osteoblastic phenotype, were expressed in AS clones before their detection in controls. Because S100A4 was not localized in the nucleus of MC3T3-E1 cells and AS clones, it is unlikely that S100A4 directly regulates the expression of these genes. Moreover, the expression of Cbfal/Osf-2 and Osx, transcription factors necessary for the expression of osteoblast-associated genes, remained unchanged in AS clones, indicating that S100A4 may be downstream to these transcription factors. These findings indicate that S100A4 is a novel negative regulator of matrix mineralization likely by modulating the process of osteoblast differentiation.

3T3 Cells↗

PubMed2003

Prostaglandin E2-mediated anabolic effect of a novel inhibitor of phosphodiesterase 4, XT-611, in the in vitro bone marrow culture.

UNLABELLED: The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells. INTRODUCTION: We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system. MATERIALS AND METHODS: Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM beta-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture. RESULTS: PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway. CONCLUSION: These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

[A clinical study on unfavorable cases of dental implant].

The clinical analysis of 462 patients (154 men and 308 women) with problems of implant therapy done at other clinics is reported. They visited the clinic for oral implant of the Tokyo Medical and Dental University Hospital during the period from January 1996 to March 2002. The results were as follows: 1) The majority of the patients (76.6%) were 40 to 69 years of age. 2) Of the total patients, 26.8% had been referred from the other outpatient clinics in our hospital, 14.7% from private dental offices and 2.6% from attending doctors. 3) There were 361 patients with complications related to the implant therapy. Seventy-six patients asked for second opinions concerned about a diagnosis or treatment recommendation. Thirty patients required maintenance of the implants. 4) The most common complications observed were peri-implantitis (184 cases). Prosthodontic complications (72 cases) included framework/resin/veneering material fractures, screw loosening and fractures. There were implant fractures in 21 cases, sensory disturbance in 20 cases, problems related to sinus in 13 cases, problems of connected teeth in 30 cases and phonetic/esthetic complications in 10 cases. 5) The types of problem implants were osseointegrated implants (196 cases), alumina-ceramic implants (74 cases), metallic blade-type implants (35 cases), pin-type implants (10 cases), subperiosteal implants (6 cases) and other implants.

Adult↗