Central Role of Pyrophosphate in Acellular Cementum Formation

Background: Inorganic pyrophosphate (PPi) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PPi are controlled by antagonistic functions of factors that decrease PPi and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PPi and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PPi in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PPi dysregulation. Results: Excess PPi in the Alpl(-/-) mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PPi in both Ank and Enpp1(-/-) mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PPi regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PPi output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PPi mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PPi inhibited mineralization and associated increases in Ank and Enpp1 mRNA. Conclusions: Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PPi, directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration

Analysis of hard tissue regeneration and Wnt signalling in dental pulp tissues after direct pulp capping with different materials

International audienceAbstract Aim To investigate the involvement of Wnt signalling and cell cycle regulation in hard tissue formation after pulp capping with several materials in a rat molar pinpoint exposure model. Methodology Thirty‐two rat molar pulps were mechanically exposed and assigned to 4 groups according to the pulp capping materials used Ca(OH) 2 , mineral trioxide aggregate (MTA), Biodentine™ and an untreated control group. After 4 weeks, the teeth were collected for microcomputed tomography to quantify reparative dentine formation. Histological analysis was then performed to evaluate the quality of the reparative dentine and the dental pulp tissue inflammatory reaction. Cyclin D1 and β‐catenin expression was examined using immunofluorescence staining. The Kruskal–Wallis followed by Dunn’s multiple comparison test was performed to determine significant differences. Results The exposed dental pulps treated with Ca(OH) 2 , MTA and Biodentine™ exhibited reparative dentine formation near the exposure site. Fibrous tissues adjacent to the exposure site were observed in the untreated group. The microcomputed tomography evaluation of MTA and Biodentine™ groups revealed significantly greater reparative dentine formation compared with the control group ( P = 0.0032 in the MTA group and P = 0.05 in the Biodentine TM group). From histological evaluations, the Biodentine TM group exhibited significantly greater reparative dentine formation grading compared with the control group ( P = 0.0152). The pulp tissues treated with Ca(OH) 2 and Biodentine™ exhibited a lower inflammatory score compared with those of the untreated control ( P = 0.0291 in the Ca(OH) 2 and P = 0.0110 in the Biodentine TM group). Ca(OH) 2 , MTA and Biodentine™ induced cyclin D1 expression in the dental pulp tissues adjacent to the reparative dentine. Moreover, the Biodentine™‐treated defects demonstrated β‐catenin expression in the pulp tissue adjacent to the newly formed reparative dentine, which was not observed with the other materials. Conclusion All test materials promoted dentine bridge formation and stimulated cyclin D1 expression. The favourable outcome after direct pulp capping with Biodentine™ involved Wnt/β‐catenin signalling. However, Wnt/β‐catenin signalling did not participate in the mechanism by which Ca(OH) 2 and MTA promoted reparative dentine formation

Decellularized extracellular matrix derived from dental pulp stem cells promotes gingival fibroblast adhesion and migration

Abstract Background Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. Results Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM–receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. Conclusions ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair

Decellularized extracellular matrix derived from dental pulp stem cells promotes gingival fibroblast adhesion and migration

International audienceBackground Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. ResultsType 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM-receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels.Conclusions ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair.</div

Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

BACKGROUND: Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. RESULTS: This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer’s research. The culture also produces glia and different sub-types of neurons. CONCLUSION: We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13041-014-0063-0) contains supplementary material, which is available to authorized users