RESUMO
PURPOSE: Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. The ability of human Dental Pulp Stem Cells (DPSCs) to differentiate into chondroblasts in vitro suggests that this stem cell type may be useful for tissue bioengineering. However, we have yet to identify a study of large animal models in which DPSCs were used to repair articular cartilage. Therefore, this study aimed to describe a novel treatment for cartilage lesion with DPSCs on a large animal model. METHODS: Mesenchymal stem cells (MSC) were obtained from deciduous teeth and characterized by flow cytometry. DPSCs were cultured and added to a collagen type I/III biomaterial composite scaffold. Brazilian miniature pig (BR-1) was used. A 6-mm diameter, full-thickness chondral defect was created in each posterior medial condyle. The defects were covered with scaffold alone or scaffold + DPSCs on the contralateral side. Animals were euthanized 6 weeks post-surgery. Cartilage defects were analyzed macroscopically and histology according to modified O'Driscoll scoring system. RESULTS: Flow cytometry confirmed characterization of DPSCs as MSCs. Macroscopic and histological findings suggested that this time period was reasonable for evaluating cartilage repair. To our knowledge, this study provides the first description of an animal model using DPSCs to study the differentiation of hyaline articular cartilage in vivo. CONCLUSION: The animals tolerated the procedure well and did not show clinical or histological rejection of the DPSCs, reinforcing the feasibility of this descriptive miniature pig model for pre-clinical studies.
Assuntos
Doenças das Cartilagens/terapia , Cartilagem Articular/crescimento & desenvolvimento , Transplante de Células-Tronco Mesenquimais , Células-Tronco/citologia , Animais , Doenças das Cartilagens/fisiopatologia , Cartilagem Articular/citologia , Diferenciação Celular/genética , Condrócitos/citologia , Condrogênese/genética , Polpa Dentária/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Suínos , Porco Miniatura , Engenharia Tecidual , Dente Decíduo/citologiaRESUMO
We have recently reported that human fallopian tubes, which are discarded during surgical procedures of women submitted to sterilization or hysterectomies, are a rich source of human fallopian tube mesenchymal stromal cells (htMSCs). It has been previously shown that human mesenchymal stromal cells may be useful in enhancing the speed of bone regeneration. This prompted us to investigate whether htMSCs might be useful for the treatment of osteoporosis or other bone diseases, since they present a pronounced capacity for osteogenic differentiation in vitro. Based on this prior knowledge, our aim was to evaluate, in vivo, the osteogenic capacity of htMSCs to regenerate bone through an already described xenotransplantation model: nonimmunosuppressed (NIS) rats with cranial defects. htMSCs were obtained from five 30-50 years old healthy women and characterized by flow cytometry and for their multipotenciality in vitro capacity (osteogenic, chondrogenic and adipogenic differentiations). Two symmetric full-thickness cranial defects on each parietal region of seven NIS rats were performed. The left side (LS) of six animals was covered with CellCeram (Scaffdex)-a bioabsorbable ceramic composite scaffold that contains 60% hydroxyapatite and 40% ß-tricalciumphosphate-only, and the right side (RS) with the CellCeram and htMSCs (10(6) cells/scaffold). The animals were euthanized at 30, 60 and 90 days postoperatively and cranial tissue samples were taken for histological analysis. After 90 days we observed neobone formation in both sides. However, in animals euthanized 30 and 60 days after the procedure, a mature bone was observed only on the side with htMSCs. PCR and immunofluorescence analysis confirmed the presence of human DNA and thus that human cells were not rejected, which further supports the imunomodulatory property of htMSCs. In conclusion, htMSCs can be used successfully to enhance bone regeneration in vivo, opening a new field for future treatments of osteoporosis and bone reconstruction.
Assuntos
Regeneração Óssea/fisiologia , Tubas Uterinas/citologia , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Transplante Heterólogo , Adulto , Animais , Diferenciação Celular , Linhagem da Célula , DNA/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Pessoa de Meia-Idade , Osteogênese , Ratos , Ratos WistarRESUMO
The present study examined the viability of bovine nuclear transferred embryos. Oocytes were matured in vitro for 17 hours and enucleated after aspiration of the first polar body and the metaphase plate. Fibroblasts from a 10-year-old Nellore cow and 45-day-old male fetus collected from slaughterhouse were used as nuclei donor. Enucleated oocytes were fused with adult and fetal fibroblasts with an electric stimulus. After electrical fusion, the couplets were incubated in TCM199 plus 10% FCS supplemented with cycloheximide and cytochalasin D for 1 hour and then cycloheximide alone for an additional 4 hours. The activated reconstructed embryos were co-cultured with granulosa cells in TCM 199 for 79 days. A total of 569 couplets were reconstructed from adult and 668 from fetal fibroblasts. After electrofusion, 181 (adult cells) and 212 (fetal cells) embryos became fused and 30 (16.6%) and 32 (15.1%) reached the blastocyst stage, respectively. After transferring 21 (adult cells) and 18 (fetal cells) blastocysts, pregnancy rates at day 90 were 19% (4) and 16.7% (3), respectively. There was no significant difference (P 0.05) between the pregnancy rates. Two pregnancies from adult cells aborted at the 4th and 5th months of gestation. One recipient that received an embryo from adult fibroblasts produced a healthy Nellore female calf weighting 40 kg. The other recipient that receive
Este trabalho examinou a viabilidade de embriões bovinos obtidos por transferência nuclear. Oócitos foram maturados in vitro por 17 horas e enucleados após a aspiração do primeiro corpúsculo polar e da placa metafásica. Fibroblastos de uma vaca Nelore de 10 anos de idade e de um feto macho de 45 dias coletado em abatedouro foram utilizados como doadores de núcleos. Os oócitos enucleados foram fusionados com fibroblastos de origem adulta e fetal após estímulo elétrico. Após fusão elétrica, as estruturas reconstruídas foram incubadas em TCM199 acrescido de 10%de SFB e suplementado com cicloheximida e citocalasina D por 1 hora e depois mais 4 horas apenas em cicloheximida. Os embriões reconstruídos e ativados foram co-cultivados em TCM199 com células da granulosa por 7-9 dias. Um total de 569 embriões foram reconstruídos com fibroblasto adulto e 668 com fibroblasto fetal. Após a eletrofusão, 181 e 212 embriões oriundos de células adultas e fetais, respectivamente, fundiram e desses 16,6% e 15,1% chegaram ao estágio de blastocisto. Após transferir 21 e 18 blastocistos oriundos de células adultas e fetais, respectivamenet, as taxas de prenhez aos 90 dias foram 19% e 16, 7%. Não houve diferença estatística (p 0,05) entre as taxas de prenhez. Duas gestações originadas de células adultas abortaram ao 4º e 5º mês de gestação. Uma receptora que recebeu embrião de fibroblasto adulto pa
RESUMO
The present study examined the viability of bovine nuclear transferred embryos. Oocytes were matured in vitro for 17 hours and enucleated after aspiration of the first polar body and the metaphase plate. Fibroblasts from a 10-year-old Nellore cow and 45-day-old male fetus collected from slaughterhouse were used as nuclei donor. Enucleated oocytes were fused with adult and fetal fibroblasts with an electric stimulus. After electrical fusion, the couplets were incubated in TCM199 plus 10% FCS supplemented with cycloheximide and cytochalasin D for 1 hour and then cycloheximide alone for an additional 4 hours. The activated reconstructed embryos were co-cultured with granulosa cells in TCM 199 for 79 days. A total of 569 couplets were reconstructed from adult and 668 from fetal fibroblasts. After electrofusion, 181 (adult cells) and 212 (fetal cells) embryos became fused and 30 (16.6%) and 32 (15.1%) reached the blastocyst stage, respectively. After transferring 21 (adult cells) and 18 (fetal cells) blastocysts, pregnancy rates at day 90 were 19% (4) and 16.7% (3), respectively. There was no significant difference (P 0.05) between the pregnancy rates. Two pregnancies from adult cells aborted at the 4th and 5th months of gestation. One recipient that received an embryo from adult fibroblasts produced a healthy Nellore female calf weighting 40 kg. The other recipient that receive
Este trabalho examinou a viabilidade de embriões bovinos obtidos por transferência nuclear. Oócitos foram maturados in vitro por 17 horas e enucleados após a aspiração do primeiro corpúsculo polar e da placa metafásica. Fibroblastos de uma vaca Nelore de 10 anos de idade e de um feto macho de 45 dias coletado em abatedouro foram utilizados como doadores de núcleos. Os oócitos enucleados foram fusionados com fibroblastos de origem adulta e fetal após estímulo elétrico. Após fusão elétrica, as estruturas reconstruídas foram incubadas em TCM199 acrescido de 10%de SFB e suplementado com cicloheximida e citocalasina D por 1 hora e depois mais 4 horas apenas em cicloheximida. Os embriões reconstruídos e ativados foram co-cultivados em TCM199 com células da granulosa por 7-9 dias. Um total de 569 embriões foram reconstruídos com fibroblasto adulto e 668 com fibroblasto fetal. Após a eletrofusão, 181 e 212 embriões oriundos de células adultas e fetais, respectivamente, fundiram e desses 16,6% e 15,1% chegaram ao estágio de blastocisto. Após transferir 21 e 18 blastocistos oriundos de células adultas e fetais, respectivamenet, as taxas de prenhez aos 90 dias foram 19% e 16, 7%. Não houve diferença estatística (p 0,05) entre as taxas de prenhez. Duas gestações originadas de células adultas abortaram ao 4º e 5º mês de gestação. Uma receptora que recebeu embrião de fibroblasto adulto pa
RESUMO
Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me(2)SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60s in final solutions of EG+DMSO1 (20% EG+20% Me(2)SO) or EG+DMSO2 (25% EG+25% Me(2)SO) or EG+GLY (25% EG+25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30s exposure of final solutions of EG+DMSO1 or EG+DMSO2 or EG+GLY. Maturation rates of 30s exposure groups were not different from the control, but 60s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG+DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG+DMSO1 (11.7%) and EG+GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.
Assuntos
Bovinos/fisiologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Oócitos/efeitos dos fármacos , Animais , Criopreservação/métodos , Crioprotetores/química , Dimetil Sulfóxido/química , Dimetil Sulfóxido/farmacologia , Etilenoglicol/química , Etilenoglicol/farmacologia , Feminino , Glicerol/química , Glicerol/farmacologia , Oócitos/citologia , Oócitos/fisiologiaRESUMO
This study evaluated in vitro maturation of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (5 microgram/ml) [DOSAGE ERROR CORRECTED]and butyrolactone I (12.5M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained, and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II revealed that hormonal treatment 2(incubation for 22 h - 10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h - 10 ng EGF/ml) presented higher repeatability percentages: TCM+ PVA (54.5% - 61/112); TCM+ pFF (65.0% - 63/97);NCSU23 + PVA (54.6% - 65/119), and NCSU23 + pFF (58.1% - 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/mland 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups.
Assuntos
Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Meiose/efeitos dos fármacos , Oócitos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Animais , Gonadotropina Coriônica/farmacologia , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária/métodos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Fertilização in vitro/veterinária , Líquido Folicular/fisiologia , Gonadotropinas Equinas/farmacologia , Microscopia de Fluorescência/veterinária , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Álcool de Polivinil/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Suínos , Fatores de TempoRESUMO
O objetivo deste trabalho foi estudar a remoção do crioprotetor, em duas ou três etapas, em embriões bovinos produzidos in vitro após a congelação em vapor de Nirtogênio. Blastocistos expandidos (1329) foram mantidos em co-cultivo (controle) ou criopreservados em 3 protocolos de congelação em vapor de nitrogênio. Os embriões foram equilibrados na solução de 10% de EG por 10 minutos e em 17%, 22% ou 28% de EG por 30 segundos. Após o envase, as palhetas foram mantidas em vapor de nitrogênio por 2 minutos e armazenadas em nitrogênio líquido. Após a descongelação, os crioprotetores foram diluídos em duas etapas, usando 0,3M de sacarose e solução isotônica ou em três etapas usando 0,3M de sacarose + 10% de EG; 0,3M de sacarose e solução isotônica. Os embriões foram co-cultivados com células da granulosa, avaliando as taxas de re-expansão após 24 horas e de eclosão após 24, 48, 72 e 96 horas. Para os grupos congelados no vapor e diluição do crioprotetor em duas etapas, as taxas de eclosão foram de 1,94; 11,88 e 6,06% para EG17, EG 22 e EG 28 respectivamente. Já para os grupos com diluição do crioprotetor em três etapas, as taxas de eclosão foram de 4,67; 9,90 e 10,78% para EG17, EG22 e EG28 respectivamente.(AU)
The aim of this study was to evaluate the dilution of cryoprotectant in 2 or steps of bovine in vitro-produced embryos after quick-freezing. A total of 1329 expanded blastocyst were kept in co-culture as control group or cryopreserved by 3 quick-freezing protocols. The embryos were exposed to 10% EG for 10 minutes then to 17%, 22% or 28% for 30 seconds. After loading, the straws were held in nitrogen vapor for 2 minutes and then plunged and stored in liquid nitrogen. After warming, cryoprotectants were diluted in two steps using 0.3 M sucrose and isotonic solution, or three steps using 0.3 M sucrose + 10% EG then 0.3 M sucrose and isotonic solution. Embryos were co-cultured on a granulosa cell monolayer and evaluated after 24 hours for re-expansion and at 24, 48, 72 and 96 hours of co-culture for hatching rates. The in vitro survival rates of embryos cryopreserved by the quick-freezing method and two-step cryoprotectant dilution were 1.94; 11.88 and 6.06%, for EG17, EG22 and EG28 groups, respectively. At the three step dilution, the in vitro survival rates were 4.67; 9.90 and 10.78% for EG17, EG22 and EG28 groups, respectively
Assuntos
Animais , Bovinos , Criopreservação/veterinária , Desenvolvimento Embrionário/fisiologia , Estruturas Embrionárias/embriologia , BovinosRESUMO
O objetivo deste trabalho foi estudar a remoção do crioprotetor, em duas ou três etapas, em embriões bovinos produzidos in vitro após a congelação em vapor de Nirtogênio. Blastocistos expandidos (1329) foram mantidos em co-cultivo (controle) ou criopreservados em 3 protocolos de congelação em vapor de nitrogênio. Os embriões foram equilibrados na solução de 10% de EG por 10 minutos e em 17%, 22% ou 28% de EG por 30 segundos. Após o envase, as palhetas foram mantidas em vapor de nitrogênio por 2 minutos e armazenadas em nitrogênio líquido. Após a descongelação, os crioprotetores foram diluídos em duas etapas, usando 0,3M de sacarose e solução isotônica ou em três etapas usando 0,3M de sacarose + 10% de EG; 0,3M de sacarose e solução isotônica. Os embriões foram co-cultivados com células da granulosa, avaliando as taxas de re-expansão após 24 horas e de eclosão após 24, 48, 72 e 96 horas. Para os grupos congelados no vapor e diluição do crioprotetor em duas etapas, as taxas de eclosão foram de 1,94; 11,88 e 6,06% para EG17, EG 22 e EG 28 respectivamente. Já para os grupos com diluição do crioprotetor em três etapas, as taxas de eclosão foram de 4,67; 9,90 e 10,78% para EG17, EG22 e EG28 respectivamente.
The aim of this study was to evaluate the dilution of cryoprotectant in 2 or steps of bovine in vitro-produced embryos after quick-freezing. A total of 1329 expanded blastocyst were kept in co-culture as control group or cryopreserved by 3 quick-freezing protocols. The embryos were exposed to 10% EG for 10 minutes then to 17%, 22% or 28% for 30 seconds. After loading, the straws were held in nitrogen vapor for 2 minutes and then plunged and stored in liquid nitrogen. After warming, cryoprotectants were diluted in two steps using 0.3 M sucrose and isotonic solution, or three steps using 0.3 M sucrose + 10% EG then 0.3 M sucrose and isotonic solution. Embryos were co-cultured on a granulosa cell monolayer and evaluated after 24 hours for re-expansion and at 24, 48, 72 and 96 hours of co-culture for hatching rates. The in vitro survival rates of embryos cryopreserved by the quick-freezing method and two-step cryoprotectant dilution were 1.94; 11.88 and 6.06%, for EG17, EG22 and EG28 groups, respectively. At the three step dilution, the in vitro survival rates were 4.67; 9.90 and 10.78% for EG17, EG22 and EG28 groups, respectively.