Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mese...Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.展开更多
AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bo...AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells(BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds(1) seeded with BMSCs;(2) wrapped with osteogenic matrix cell sheets; or(3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmentalor continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone nonunion. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness(mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.展开更多
AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential mediu...AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.展开更多
We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation ...We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.展开更多
AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varyi...AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varying oxygen conditions: Normoxia(21% O_2) for 14 d(NN), normoxia for 7 d followed by hypoxia(5% O_2) for 7 d(NH), hypoxia for 7 d followed by normoxia for 7 d(HN), or hypoxia for 14 d(HH). Osteogenesis was evaluated by observing changes in cell morphology and calcium deposition, and by measuring osteocalcin secretion(ELISA) and calcium content. In vivo syngeneic transplantation using OMCSs and β-tricalcium phosphate discs, preconditioned under NN or HN conditions, was also evaluated by histology, calcium content measurements,and real-time quantitative PCR.RESULTS In the NN and HN groups, differentiated, cuboidal-shaped cells were readily observed, along with calcium deposits. In the HN group, the levels of secreted osteocalcin increased rapidly from day 10 as compared with the other groups, and plateaued at day 12(P < 0.05). At day 14, the HN group showed the highest amount of calcium deposition. In vivo, the HN group showed histologically prominent new bone formation, increased calcium deposition, and higher collagen type Ⅰ?messenger RNA expression as compared with the NN group.CONCLUSION The results of this study indicate that modifying oxygen tension is an effective method to enhance the osteogenic ability of MSCs used for OMCSs.展开更多
Background: Japanese emergency medical services (EMS) can be used by anyone for free. Recently, EMS usage has increased;the increased costs and the prolonged time for ambulance transport have become recent social prob...Background: Japanese emergency medical services (EMS) can be used by anyone for free. Recently, EMS usage has increased;the increased costs and the prolonged time for ambulance transport have become recent social problems. Objective: We surveyed the willingness to pay (WTP) for resuscitation provided by EMS. Methods: In November 2011, men and women (3160) aged 20 - 59 years were asked to assume that they were experiencing a cardiopulmonary arrest (CPA), and their WTP for EMS services was assessed in the following three situations: Case A, ambulance transport alone;Case B, chest compression in addition to ambulance transport;and Case C, artificial ventilation through chest compression and intratracheal intubation, in addition to ambulance transport. We calculated the mean WTP for each case. Results: The WTP for Case A, B, and C were ￥6,696 ($65.0), ￥16,081 ($156.1), and ￥27,505 ($267.0), respectively. The WTP for Case B was significantly higher in respondents aged 40 - 59 years compared to those aged 20 - 39 years. The WTP for case B and C were significantly higher in males compared to females. WTP was significantly lower in students than it was in private employees. Although women’s intention to pay was higher than that of men, their WTP was lower than that of men. Public employees’ and students’ intention to pay was significantly lower than that of private employees. Conclusions: Our study provides information about the optimal fee for EMS, which will be useful for discussions on the feasibility of introducing a fee for EMS in Japan.展开更多
Objective: To determine the effect of family smoking habits on adolescent smoking experience for information for anti-smoking strategies. Subjects and Methods: We analyzed data from 4776 junior high school students an...Objective: To determine the effect of family smoking habits on adolescent smoking experience for information for anti-smoking strategies. Subjects and Methods: We analyzed data from 4776 junior high school students and 5047 high school students responding to the “Survey on Prevention of Life-Style Related Diseases of Children” conducted in Nara Prefecture of Japan in 2004. The chi-square test determined the odds ratios (ORs) for smoking experience rates of the two groups of students according to the smoking habits of their fathers, mothers, older brothers, and older sisters. Logistic regression analysis was also performed for smoking experience according to school year, sex, and family smoking habit. Results: The OR for smoking in junior high school students with a father as the only smoking family member against all other cases was 0.60, and the OR with a mother as the only smoking family member was 2.50. The OR with a smoking father against cases with no smoking family members was 1.23, and the OR with a smoking mother against cases with no smoking family members was 3.50. The OR for smoking in high school students with a father as the only smoking family member against all other cases was 0.76, and the OR with a mother as the only smoking family member was 2.66. The OR with a smoking father against cases with no smoking family members was 1.59, and the OR with a smoking mother against cases with no smoking family member was 3.26. All these ORs showed significance. Conclusion: Maternal smoking was associated with a particularly high risk of smoking experience in junior high and high school students. Prevention of maternal smoking should be given high priority to reduce smoking rates in adolescents.展开更多
文摘Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.
基金Supported by Grant-in-Aid for Young Scientists(KAKENHI)
文摘AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells(BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds(1) seeded with BMSCs;(2) wrapped with osteogenic matrix cell sheets; or(3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmentalor continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone nonunion. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness(mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.
基金Supported by Grant-in-Aid for scientific research from the Ministry of Health,Labour and Welfare,Japan
文摘AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.
文摘We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.
文摘AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varying oxygen conditions: Normoxia(21% O_2) for 14 d(NN), normoxia for 7 d followed by hypoxia(5% O_2) for 7 d(NH), hypoxia for 7 d followed by normoxia for 7 d(HN), or hypoxia for 14 d(HH). Osteogenesis was evaluated by observing changes in cell morphology and calcium deposition, and by measuring osteocalcin secretion(ELISA) and calcium content. In vivo syngeneic transplantation using OMCSs and β-tricalcium phosphate discs, preconditioned under NN or HN conditions, was also evaluated by histology, calcium content measurements,and real-time quantitative PCR.RESULTS In the NN and HN groups, differentiated, cuboidal-shaped cells were readily observed, along with calcium deposits. In the HN group, the levels of secreted osteocalcin increased rapidly from day 10 as compared with the other groups, and plateaued at day 12(P < 0.05). At day 14, the HN group showed the highest amount of calcium deposition. In vivo, the HN group showed histologically prominent new bone formation, increased calcium deposition, and higher collagen type Ⅰ?messenger RNA expression as compared with the NN group.CONCLUSION The results of this study indicate that modifying oxygen tension is an effective method to enhance the osteogenic ability of MSCs used for OMCSs.
文摘Background: Japanese emergency medical services (EMS) can be used by anyone for free. Recently, EMS usage has increased;the increased costs and the prolonged time for ambulance transport have become recent social problems. Objective: We surveyed the willingness to pay (WTP) for resuscitation provided by EMS. Methods: In November 2011, men and women (3160) aged 20 - 59 years were asked to assume that they were experiencing a cardiopulmonary arrest (CPA), and their WTP for EMS services was assessed in the following three situations: Case A, ambulance transport alone;Case B, chest compression in addition to ambulance transport;and Case C, artificial ventilation through chest compression and intratracheal intubation, in addition to ambulance transport. We calculated the mean WTP for each case. Results: The WTP for Case A, B, and C were ￥6,696 ($65.0), ￥16,081 ($156.1), and ￥27,505 ($267.0), respectively. The WTP for Case B was significantly higher in respondents aged 40 - 59 years compared to those aged 20 - 39 years. The WTP for case B and C were significantly higher in males compared to females. WTP was significantly lower in students than it was in private employees. Although women’s intention to pay was higher than that of men, their WTP was lower than that of men. Public employees’ and students’ intention to pay was significantly lower than that of private employees. Conclusions: Our study provides information about the optimal fee for EMS, which will be useful for discussions on the feasibility of introducing a fee for EMS in Japan.
文摘Objective: To determine the effect of family smoking habits on adolescent smoking experience for information for anti-smoking strategies. Subjects and Methods: We analyzed data from 4776 junior high school students and 5047 high school students responding to the “Survey on Prevention of Life-Style Related Diseases of Children” conducted in Nara Prefecture of Japan in 2004. The chi-square test determined the odds ratios (ORs) for smoking experience rates of the two groups of students according to the smoking habits of their fathers, mothers, older brothers, and older sisters. Logistic regression analysis was also performed for smoking experience according to school year, sex, and family smoking habit. Results: The OR for smoking in junior high school students with a father as the only smoking family member against all other cases was 0.60, and the OR with a mother as the only smoking family member was 2.50. The OR with a smoking father against cases with no smoking family members was 1.23, and the OR with a smoking mother against cases with no smoking family members was 3.50. The OR for smoking in high school students with a father as the only smoking family member against all other cases was 0.76, and the OR with a mother as the only smoking family member was 2.66. The OR with a smoking father against cases with no smoking family members was 1.59, and the OR with a smoking mother against cases with no smoking family member was 3.26. All these ORs showed significance. Conclusion: Maternal smoking was associated with a particularly high risk of smoking experience in junior high and high school students. Prevention of maternal smoking should be given high priority to reduce smoking rates in adolescents.
