Ingénierie tissulaire de la pulpe dentaire : vers le développement d’un médicament de thérapie innovante

Dental research currently explores the potential of cell-based products and tissue engineering protocols to be used as alternatives to usual pulp/dentin and bone therapies. In this context, stem/progenitor cells appear to be particularly appropriate because of their high expansion ability and differentiation potential both in vitro and in vivo. If bone marrow and adipose tissue are considered potential sources of stem/progenitor cells, painful collection protocols, the decline of the amount of stem/ progenitor cells with age, the necessity of general anesthesia, reduced proliferation capacity, and risk of morbidity at the collection site encourage the search for alternative candidates. Human impacted third molars are frequently removed for therapeutic reasons and the loose connective tissue they contain, the dental pulp, appears to be a valuable source of stem/progenitor cells for pulp/dentin and bone engineering. Indeed, it contains various cell populations that exhibit osteo/odontoblastic differentiation capabilities and that can be cryopreserved for periods of time greater than 6 months. Interestingly, human dental pulp cell (HDPC) populations were recently successfully used for regenerating human pulp/dentin and bone. Cell-based products for tissue engineering are now referred to as human cellular tissue-based products or advanced therapy medicinal products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate cell production. These ‘‘good manufacturing practices’’ include recommendations regarding laboratory cell culture procedures to ensure optimal reproducibility, efficacy, and safety of the final medicinal productCes dernières années, des thérapies à base de cellules mésenchymateuses ont été développées pour améliorer les thérapies qui visent à réparer l'homme et notamment la pulpe dentaire. Dans ce contexte, la dent apparait comme la source de cellules mésenchymateuses, souches ou progénitrices, permettant de réparer la pulpe dentaire. En effet, la pulpe dentaire est facile d'accès et les cellules pulpaires présentent un fort potentiel de différenciation. Actuellement, les différents organismes de contrôle recommandent d'utiliser des procédures standardisées pour l'isolement, le stockage et l'expansion des cellules en culture pour garantir une sécurité et une reproductibilité optimale lorsque les cellules sont utilisées en culture cellulaire. Cependant, la plupart des procédures utilisées pour la production de cellules à partir de la pulpe dentaire ne sont pas entièrement satisfaisante, car elles peuvent altérer les propriétés biologiques et la qualité des cellules. En effet, les procédures d'isolement cellulaire, d'enrichissement, de cryopréservation et d'amplification pendant de nombreux passages dans des milieux contenant des produits d'origine animale ou humaine sont connues pour affecter le phénotype des cellules, la viabilité, la prolifération et les capacités de différenciation. Ce travail de thèse s'intéresse à compiler les stratégies actuelles de fabrication de produits cellulaires à partir de la pulpe dentaire, puis il propose de nouveaux protocoles pour améliorer l'efficacité, la reproductibilité et la sécurité de ces nouvelles stratégies thérapeutiques. Ainsi nous avons isolé, amplifié et cryopréservé des cellules de la pulpe dentaire. Grace à un travail d'immunophénotypage, nous avons pu étudier différentes souspopulations à l'intérieur de la population totale. Enfin nous avons montré que ces cellules sont capables de rester congelées pendant plus de 500 jours sans présenter d'anomalies du caryotype et de conserver un potentiel de différenciation ostéo/odontogéniqu

A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton’s jelly samples

Transplantation of mesenchymal stem/stromal cells (MSCs) has emerged as an effective method to treat diseased or damaged organs and tissues, and hundreds of clinical trials using MSCs are currently under way to demonstrate the validity of such a therapeutic approach. However, most MSCs used for clinical trials are prepared in research laboratories with insufficient manufacturing quality control.In particular, laboratories lack standardized procedures for in vitro isolation of MSCs from tissue samples, resulting in heterogeneous populations of cells and variable experimental and clinical results. MSCs are now referred to as Human Cellular Tissue-based Products or Advanced Therapy Medicinal Products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and from the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate production of these cells. These guidelines, commonly called “Good Manufacturing Practices” (GMP), include recommendations about laboratory cell culture procedures to ensure optimal reproducibility, efficacy and safety of the final medicinal product. In particular, the Food and Drug Administration divides ex vivo cultured cells into “minimally” and “more than minimally” manipulated samples, in function of the use or not of procedures “that might alter the biological features of the cells”. Today, minimal manipulation conditions have not been defined for the collection and isolation of MSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have been reported so far are unsatisfactory, because of the use of xeno- or allogeneic cell culture media, enzymatic treatment and long-term cell amplification that are known to alter the quality of MSCs. The aim of this study was to describe a standardized procedure for recovering MSCs with minimal handling from two promising sources, the dental pulp (DP) and the Wharton’s jelly (WJ) of the umbilical cord. The quality and homogeneity of the expanded cell populations were assessed by using flow cytometry with criteria that go beyond the International Society of Cellular Therapy (ISCT) guidelines for MSC characterization

Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

International audienceMesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31 − cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31 − DP cell population contained 1.4% of CD56 + , 1.5% of CD146 + , 2.4% of CD271 + and 6.3% of MSCA-1 + cells but very few Stro-1 + cells (≤1%). CD56 + , CD146 + , CD271 + , and MSCA-1 + cell subpopulations expressed various levels of these markers. CD146 + MSCA-1 + , CD271 + MSCA-1 + , and CD146 + CD271 + cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56 + , CD271 + , MSCA-1 + , and Stro-1 + cells, and MSCA-1 by 15-30% of CD56 + , CD146 + , CD271 + , and Stro-1 + cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56 + cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146 + CD56 + , MSCA-1 + CD56 + , and CD146 + MSCA-1 + cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

Dental pulp tissue engineering : toward the development of a cell-based medicinal product

Ces dernières années, des thérapies à base de cellules mésenchymateuses ont été développées pour améliorer les thérapies qui visent à réparer l'homme et notamment la pulpe dentaire. Dans ce contexte, la dent apparait comme la source de cellules mésenchymateuses, souches ou progénitrices, permettant de réparer la pulpe dentaire. En effet, la pulpe dentaire est facile d'accès et les cellules pulpaires présentent un fort potentiel de différenciation. Actuellement, les différents organismes de contrôle recommandent d'utiliser des procédures standardisées pour l'isolement, le stockage et l'expansion des cellules en culture pour garantir une sécurité et une reproductibilité optimale lorsque les cellules sont utilisées en culture cellulaire. Cependant, la plupart des procédures utilisées pour la production de cellules à partir de la pulpe dentaire ne sont pas entièrement satisfaisante, car elles peuvent altérer les propriétés biologiques et la qualité des cellules. En effet, les procédures d'isolement cellulaire, d'enrichissement, de cryopréservation et d'amplification pendant de nombreux passages dans des milieux contenant des produits d'origine animale ou humaine sont connues pour affecter le phénotype des cellules, la viabilité, la prolifération et les capacités de différenciation. Ce travail de thèse s'intéresse à compiler les stratégies actuelles de fabrication de produits cellulaires à partir de la pulpe dentaire, puis il propose de nouveaux protocoles pour améliorer l'efficacité, la reproductibilité et la sécurité de ces nouvelles stratégies thérapeutiques. Ainsi nous avons isolé, amplifié et cryopréservé des cellules de la pulpe dentaire. Grace à un travail d'immunophénotypage, nous avons pu étudier différentes souspopulations à l'intérieur de la population totale. Enfin nous avons montré que ces cellules sont capables de rester congelées pendant plus de 500 jours sans présenter d'anomalies du caryotype et de conserver un potentiel de différenciation ostéo/odontogéniqueDental research currently explores the potential of cell-based products and tissue engineering protocols to be used as alternatives to usual pulp/dentin and bone therapies. In this context, stem/progenitor cells appear to be particularly appropriate because of their high expansion ability and differentiation potential both in vitro and in vivo. If bone marrow and adipose tissue are considered potential sources of stem/progenitor cells, painful collection protocols, the decline of the amount of stem/ progenitor cells with age, the necessity of general anesthesia, reduced proliferation capacity, and risk of morbidity at the collection site encourage the search for alternative candidates. Human impacted third molars are frequently removed for therapeutic reasons and the loose connective tissue they contain, the dental pulp, appears to be a valuable source of stem/progenitor cells for pulp/dentin and bone engineering. Indeed, it contains various cell populations that exhibit osteo/odontoblastic differentiation capabilities and that can be cryopreserved for periods of time greater than 6 months. Interestingly, human dental pulp cell (HDPC) populations were recently successfully used for regenerating human pulp/dentin and bone. Cell-based products for tissue engineering are now referred to as human cellular tissue-based products or advanced therapy medicinal products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate cell production. These ‘‘good manufacturing practices’’ include recommendations regarding laboratory cell culture procedures to ensure optimal reproducibility, efficacy, and safety of the final medicinal produc

Approche didactique de l'urgence en odontologie prothétique (proposition de deux arbres décisionnels)

LYON1-BU Santé Odontologie (693882213) / SudocSudocFranceF

Le conditionnement gingival mécanico-chimique (à propos d'une étude)

LYON1-BU Santé Odontologie (693882213) / SudocSudocFranceF

Les "cellules souches dentaires" (exemple des cellules souches pulpaires et applications possibles en Odontologie)

LYON1-BU Santé Odontologie (693882213) / SudocSudocFranceF

Evolution temporelle de la diversité génétique neutre et adaptative de la population de saumon atlantique de l’Allier

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Immunophenotyping reveals the diversity of human dental pulp mesenchymal stromal cells in vivo and their evolution upon in vitro amplification

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31- cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31- DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤1%). CD56+, CD146+, CD271+ and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+ and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+ and Stro-1+ cells, and MSCA-1 by 15-30% of CD56+, CD146+, CD271+ and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by less than 1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+ and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

Evolution temporelle de la diversité génétique neutre et adaptative de la population de saumon atlantique de l’Allier

absen