ES cells-derived ectomesenchymal cells for tooth engineering.

Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells. Les progrès en matière de biologie de cellules souches et d’ingénierie tissulaire permettent d’envisager le développement de nouvelles thérapies pour pallier les pertes de tissus dentaires consécutives à des traumatismes, des situations pathologiques ou au vieillissement. La possibilité de générer une dent en mimant le développement, par réassociations entre cellules dentaires épithéliales et mésenchymateuses dérivées des crêtes neurales (CN), a été démontrée chez la souris. Dans la recherche de ressources cellulaires utilisables pour un transfert chez l’homme, les cellules souches pluripotentes pourraient constituer une alternative. Notre but est d’obtenir à partir de ces dernières, des cellules ectomésenchymateuses capables d’interagir avec un épithélium dentaire pour promouvoir l’histomorphogenèse d’une dent. Pour cela, deux protocoles de différenciation de cellules ES, utilisant la cyclopamine ou une combinaison de FGF2/BMP4, ont été mis au point. Les cellules induites ont été caractérisées par PCR quantitative. Les deux protocoles de différenciation amènent les cellules à acquérir en 10 jours, une morphologie de type mésenchymateux. Après induction, l’expression des gènes de pluripotence chute de façon drastique alors que celle des gènes spécificateurs de CN est activée. Toutefois, la cinétique varie selon le protocole. Le gène Twist, dont l’expression dans les CN est associée à un engagement vers l’ectomésenchyme, n’est activé significativement que sous l’action de FGF2/BMP4. Des expériences de réassociations avec un épithélium dentaire sont en cours pour évaluer le potentiel odontogène des cellules ES différenciées. A terme, ces approches menées chez la souris devraient permettre de définir une stratégie pour l’obtention de cellules compétentes humaines.Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells. Les progrès en matière de biologie de cellules souches et d’ingénierie tissulaire permettent d’envisager le développement de nouvelles thérapies pour pallier les pertes de tissus dentaires consécutives à des traumatismes, des situations pathologiques ou au vieillissement. La possibilité de générer une dent en mimant le développement, par réassociations entre cellules dentaires épithéliales et mésenchymateuses dérivées des crêtes neurales (CN), a été démontrée chez la souris. Dans la recherche de ressources cellulaires utilisables pour un transfert chez l’homme, les cellules souches pluripotentes pourraient constituer une alternative. Notre but est d’obtenir à partir de ces dernières, des cellules ectomésenchymateuses capables d’interagir avec un épithélium dentaire pour promouvoir l’histomorphogenèse d’une dent. Pour cela, deux protocoles de différenciation de cellules ES, utilisant la cyclopamine ou une combinaison de FGF2/BMP4, ont été mis au point. Les cellules induites ont été caractérisées par PCR quantitative. Les deux protocoles de différenciation amènent les cellules à acquérir en 10 jours, une morphologie de type mésenchymateux. Après induction, l’expression des gènes de pluripotence chute de façon drastique alors que celle des gènes spécificateurs de CN est activée. Toutefois, la cinétique varie selon le protocole. Le gène Twist, dont l’expression dans les CN est associée à un engagement vers l’ectomésenchyme, n’est activé significativement que sous l’action de FGF2/BMP4. Des expériences de réassociations avec un épithélium dentaire sont en cours pour évaluer le potentiel odontogène des cellules ES différenciées. A terme, ces approches menées chez la souris devraient permettre de définir une stratégie pour l’obtention de cellules compétentes humaines

Differences between normal and demineralized dentine pretreated with silver fluoride and potassium iodide after an in vitro challenge by Streptococcus mutans

The document attached has been archived with permission from the Australian Dental Association (8 March 2008). An external link to the publisher’s copy is included.Background: The application of diamine silver fluoride (Ag(NH3)2F) and potassium iodide (KI) to demineralized dentine has been shown to inhibit the growth of Streptococcus mutans. The purpose of this study was to observe the differences between demineralized and non-demineralized dentine treated with AgF/KI. Methods: Thirty-five dentine discs were bonded to the bases of 5mL polycarbonate screw top vials which were filled with nutrient medium, sterilized and placed into the overflow from a continuous culture of S. mutans. Samples were divided as follows: 10 samples of demineralized dentine; 10 samples of demineralized dentine treated with AgF/KI; 5 samples of non-demineralized dentine; and 10 samples of non-demineralized dentine treated with AgF/KI. Following two weeks connected to the Chemostat, an electron probe microanalysis (EPMA) of percentage weights and penetration depths of calcium, phosphorous silver and fluoride was conducted. Bacterial growth was monitored by taking optical density readings of the growth medium in each vial and outer surfaces of the specimens were examined by scanning electron microscopy (SEM). Results: AgF/KI treatment of demineralized and non-demineralized dentine prevented biofilm formation and reduced further demineralization by S. mutans. AgF/KI treatment of demineralized dentine was more effective in reducing dentine breakdown and the growth of S. mutans. Significantly higher levels of silver and fluoride were deposited within demineralized dentine. Conclusions: A topical treatment with AgF/KI on dentine reduced in vitro caries development and inhibited surface biofilm formation. Reduction of in vitro caries development and viability of S. mutans was more pronounced on the dentine samples that had been demineralized prior to the application of AgF/KI.GM Knight, JM McIntyre, GG Craig, Mulyani, PS Zilm and NJ Gull

Review of Matrix Metalloproteinases’ Effect on the Hybrid Dentin Bond Layer Stability and Chlorhexidine Clinical Use to Prevent Bond Failure

This review describes the relationship between dentin collagen hybrid bond layer degradation and the Matrix Metalloproteinases (MMPs) after their release by acid etch and rinse adhesives and self etching bonding adhesives that can reduce the bond stability over time. MMP-2, MMP-8 and MMP-9 are indicated as the active proteases that breakdown the collagen fibrils in the hybrid bond layer. Phosphoric acid in the acid etch and rinse bonding process and acid primers in the self etch process are implicated in the release of these proteases and their activation by several non-collagen proteins also released from dentin by the etching. MMPs are released in saliva by salivary glands, by cells in the gingival crevices to crevicular fluid and by pulpal odontoblasts cells to the dentinal fluids. These sources may affect the hybrid layer also. Evidence of the bond strength deterioration over time and the ability of Chlorhexidine to prevent bond deterioration by inhibiting MMP action are discussed. Dentin Bonding procedure utilizing Chlorhexidine for different application times and concentrations are being developed. The application of 2% Chlorhexidine to the phosphoric acid etch surface after rinsing off the acid is the only procedure that has been clinically tested for a longer period of time and shown to prevent bond strength degradation so far. The adoption of this procedure is recommended as means of improving bond stability at this time

Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations

Background: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. Methods: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. Results: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. Conclusions: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations

The role of matrix metalloproteinases (MMPs) in human caries

The objective of this review is to summarize our understanding of the role of host matrix metalloproteinases (MMPs) in the caries process and to discuss new therapeutic avenues. MMPs hydrolyze components of the extracellular matrix and play a central role in many biological and pathological processes. MMPs have been suggested to play an important role in the destruction of dentin organic matrix following demineralization by bacterial acids and, therefore, in the control or progression of carious decay. Host-derived MMPs can originate both from saliva and from dentin. They may be activated by an acidic pH brought about by lactate release from cariogenic bacteria. Once activated, they are able to digest demineralized dentin matrix after pH neutralization by salivary buffers. Furthermore, the degradation of SIBLINGs (Small Integrin-binding Ligand N-linked Glycoproteins) by the caries process may potentially enhance the release of MMPs and their activation. This review also explores the different available MMP inhibitors, natural or synthetic, and suggests that MMP inhibition by several inhibitors, particularly by natural substances, could provide a potential therapeutic pathway to limit caries progression in dentin