Main description:

This volume provides methods and approaches to study genetic and environmental regulatory controls on odontogenesis. Chapters guide readers through protocols for isolation and characterization of both epithelial and mesenchymal dental cells, methods on isolation, phenotypic characterization, expansion, differentiation, immunofluorescence, in situ hybridization, immunohistochemistry, imaging protocols, rodent dental fluorosis model, 3D assessment of crown size, dental diseases models, next generation sequencing, genetic and epigenetic studies, genome-wide association studies as well as clinical protocols for measurement of early childhood caries and saliva, and supragingival fluids and biofilm collection and subsequent analyses. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Odontogenesis: Methods and Protocols aims to guide researchers towards elucidating the secrets and mysteries of a fascinating and unique organ, the tooth.

Contents:

Part I: Protocols for Establishment of Dental Cells Lines and Animal Models

1. Micro-dissection and Isolation of Mouse Dental Epithelial Cells of Continuously Growing Mouse Incisors

Hidemitsu Harada and Keishi Otsu

2. Establishment of an Immortalized Mouse Bmp2 Knock--Out Dental Papilla Mesenchymal Cell Line Wen'an Xu and Shuo Chen

3. Establishment of Stable Cell Lines from Primary Human Dental Pulp Stem Cells

Elizabeth Guirado, Youbin Zhang, and Anne George

4. Isolation of Dental Stem Cell Enriched Populations from Continuously Growing Mouse Incisors

Anamaria Balic

5. Application of Cell Lineage Tracing Combined with Immunofluorescence in the Study of Dentogenesis

Yan Jing, Chaoyuan Li, and Jian Q. Feng

6. Tissue Recombination and Kidney Capsule Transplantation Assays for the Study of Epithelial-mesenchymal Interactions

Lucia Jimenez-Rojo and Thimios Mitsiadis

7. Dental Mesenchymal Stem Cells: Dental Pulp Stem Cells, Periodontal Ligament Stem Cells, Apical Papilla Stem Cells, Primary Teeth Stem Cells: Isolation, Characterization and Expansion for Tissue Engineering

Mey Al-Habib and George T.-J. Huang

Part II: Protocols for Dental Stem Cells and Tissue Regeneration

8. Phenotypic Identification of Dental Pulp Mesenchymal Stem/Stromal Cells Subpopulations with Multiparametric Flow Cytometry

Maxime Ducret, Jean-Christophe Farges, Marielle Pasdeloup, Emeline Perrier-Groult, Andreas Mueller, Frederic Mallein-Gerin, and Hugo Fabre

9. Dental Pulp Stem Cells: Isolation, Characterization, Expansion and Odontoblast Differentiation for Tissue Engineering

Qing, Dong; Wang, Yuanyuan; Mohabatpour, Fatemeh; Zheng, Li; Papagerakis, Silvana; Chen, Daniel; and Papagerakis, Petros

10. In vitro Analysis of Intramolecular Signaling Events in PDLSCs using Confocal and TIRF Microscopy

Annette Merkel and Anne George

11. A Mouse Model to Study Reparative Dentinogenesis

Babb RC, Chandrasekaran D, Zaugg LK, and Sharpe PT

12. Multiwalled Carbon Nanotubes for Dental Applications

Petros Kechagioglou;Eleftherios Andriotis; Petros Papagerakis; and Silvana Papagerakis

13. Peptide-mediated Biomimetic Regrowth of Human Enamel in situ

Kaushik Mukherjee, Qichao Ruan, and Janet Moradian-Oldak

14. Bioengineering Tooth Bud Constructs using GelMA Hydrogel Elizabeth E. Smith and Pamela C. Yelick

15. Whole-mount In Situ Hybridization of Mouse Embryos Using DIG-labeled RNA Probes

Jingyi Wu and Xiaofang Wang

Part III: Protocols for Studying Gene and Protein Expression

16. In Situ Hybridization on Mouse Paraffin Sections Using DIG-labeled RNA Probes

Jingyi Wu, Jian Q. Feng, and Xiaofang Wang

17. Methods for in situ Proteins Visualization in Dental Mineralized Tissues

D. Hotton, A. Berdal, and A. Bolanos

18. In Situ Hibridization in Mineralized Tissues: The Added Value of LNA Probes for RNA Detection

Lignon G, Hotton D, Berdal A, and Bolanos A

19. Immunofluorescence Procedure for Developing Enamel Tissues

Xu Yang and Elia Beniash

20. Silver-albumin Tissue Staining Protocol to Visualize Odontogenesis in Whole Embryos

Julia C. Boughner and David M. L. Cooper

21. Isolation of SIBLING Proteins from Bone and Dentin Matrix

Jingyi Wu and Xiaofang Wang

22. Immunohistochemical Co-localization of Amelogenin and Ameloblastin in Developing Enamel Matrix

Rucha Arun Bapat and Janet Moradian-Oldak

23. The Expression and Purification of Recombinant Mouse Ameloblastin in e-coli

Jingtan Su, Rucha Bapat, and Janet Moradian-Oldak

Part IV: Protocols for Biochemistry and Imaging

24. Protocols for Studying Formation and Mineralization of Dental Tissues in vivo

Extraction Protocol for Isolating Dentin Matrix Proteins from Developing Teeth

Yasuo Yamakoshi, Jan C-C Hu, Mari M. Saito, and James P. Simmer

25. Purification of Developing Enamel Matrix Proteins using Preparative SDS PAGE

Steven J. Brookes and Claire M. Gabe

26. Using ImageJ (FIJI) to Analyse and Present X Ray CT Images of Enamel

Steven J. Brookes

27. Scanning Electron Microscopy (SEM) Methods for Dental Enamel

Steinar Risnes, Muhammad Saeed, and Amer Sehic

28. Microcomputed Tomography Imaging in Odontogenesis Studies

Kostas Verdelis and Phil Salmon

29. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) for the Examination of Dental Hard Tissues

Victor E. Arana-Chavez and Leticia S. Castro-Filice

30. Rodent Dental Fluorosis Model--Extraction of Enamel Organ from Rat Incisors

M. Suzuki and J. D. Bartlett

Part V: Protocols to Study Dental Diseases

31. Three Dimensional Assessment of Crown Size and Eruption Space for Developing Third Molars: Data Collection Techniques Based on Cone-beam Computed Tomography (CBCT)

D.F. Marchiori, G.V. Packota, and J.C. Boughner

32. Protocols to Study Dental Caries In Vitro: Microbial Caries Models

Bennett T. Amaechi, Livia M. A. Tenuta, Antonio P. Ricomini Filho, and Jaime A. Cury

33. In vitro Caries Models for the Assessment of Novel Restorative Materials

Sulaiman Ghandourah, Basma; Lefkelidou, Anna;Said, Raed; Chatzistavrou, Xanthippi; Flannagan, Susan; Gonzales-Cabezas, Carlos; Fenno, Christopher J.; Zheng, Li;Papagerakis, Silvana; and Papagerakis, Petros

34. Protocols to Study Dental Caries In Vitro: pH Cycling Models

Bennett T. Amaechi

35. In Vivo Rodent Models for Studying Dental Caries and Pulp Disease

Hsiao, June; Wang, Yuanyuan;Liu, Ruirui; Zheng, Li; Said; Raed; Hadjiyski, Lubomir; Cha, Heekon; Botero, Tatiana; Chatzistavrou, Xanthippi; Qing, Dong;Papagerakis, Silvana; Papagerakis, Petros

36. Protocol GenoDENT: Implementation of a New NGS Panel for Molecular Diagnosis of Genetic Disorders with Orodental Involvement

Tristan Rey, Julien Tarabeux, Benedicte Gerard, Marion Delbarre, Antony Le Bechec Corinne Stoetzel, Megana Prasad, Virginie Laugel-Haushalter, Marzena Kawczynski, Jean Muller, Jamel Chelly, Helene Dollfus, Marie-Cecile Maniere, and Agnes Bloch-Zupan

Part VI: Protocols for Genetic, Epigenetic and Clinical Studies

37. Protocols for Genetic and Epigenetic Studies of Rare Diseases Affecting Dental Tissues

Bruna Rabelo Amorim, Pollyanna Almeida Costa dos Santos, Caroline Lourenco de Lima, Denise Carleto Andia, Juliana Forte Mazzeu, and Ana Carolina Acevedo

38. Protocols, Methods, and Tools for Genome-wide Association Studies (GWAS) of Dental Traits

Cary S. Agler, Dmitry Shungin, Andrea G. Ferreira Zandona, Paige Schmadeke,Patricia V. Basta, Jason Luo, John Cantrell,Thomas D. Pahel Jr., Beau Meyer, John R. Shaffer, Arne S. Schaefer, Kari E. North, and Kimon Divaris

39. Measurement of Early Childhood Oral Health for Research Purposes: Dental Caries Experience and Developmental Defects of the Enamel in the Primary Dentition

Jeannie Ginnis, Andrea G. Ferreira Zandona, Gary D. Slade, John Cantrell, Mikafui E. Antonio,Bhavna T. Pahel, Beau D. Meyer, and Kimon Divaris

40. The Supragingival Biofilm in Early Childhood Caries: Clinical and Laboratory Protocols and Bioinformatics Pipelines Supporting Oral Metagenomics, Metatranscriptomics and Metabolomics Studies of the Oral Microbiome

Kimon Divaris, Dmitry Shungin,Adaris Rodriguez-Cortes, Patricia V. Basta, Jeff Roach,Andrea G. Ferreira Zandona, Jeannie Ginnis, Sivapriya Ramamoorthy,Jakub Kwintkiewicz, Natasha Butz, and M. Andrea Azcarate-Peril

41. Saliva and Gingival Crevicular Fluid (GCF) Collection for Biomarkers Screening

Petros Papagerakis,Raed Said, Doohak Kim, Amber A. Ehlert, Kevin K. M. Chung, and Silvana Papagerakis