Massachusetts Institute of Technology Harvard Medical School Brigham and Women’s/Massachusetts General Hosp. VA Boston Healthcare System 2.79J/3.96J/BE.441/HST522J DENTAL TISSUE REPLACEMENT AND REGENERATION M. Spector, Ph.D. and I.V. Yannas, Ph.D. Image source: OCW Dental Implant Designs and Materials Sapphire Alumina Titanium Carbon Images removed due to copyright considerations. Alumina Alumina Carbon Blade Implant Images removed due to copyright considerations. “Commercially pure” Titanium Two-Stage Design; to shield the artificial root from loading during the initial stage of healing Images removed due to copyright considerations. Dental Implant Designs and Materials Hydroxyapatite- Coated Implants Images removed due to copyright considerations. Secondary dentin Dental Caries Osteodentin Calcium hydroxide liner Secondary dentin Tissue Engineering Enamel Regeneration Dentin Regeneration Pulp Regeneration Restorative Materials Ceramics: porcelain Metals: amalgam, Implants Polymer: composite, gutta percha ELEMENTS FOR TISSUE REGENERATION/ENGINEERING CELLS Autologous, Adult (pulp and bone marrow stromal stem cell) MATRIX Collagen-GAG CYTOKINES Regulation of phenotype Matrix biosynthesis Tissue Engineering of Complex Tooth Structures on Biodegradable Polymer Scaffolds ? Cells dissociated from porcine third molar tooth buds. ? Cells seeded onto PLA fiber mesh and implanted in rats for 20 to 30 wks. ? Resulting tooth structures contained dentin (odontoblasts), a well-defined pulp chamber, putative cementoblasts, and a morphologically correct enamel. ? Results suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues. C.S. Young, et al. J Dent Res 81(10): 695-700, 2002 Growth of Porcine Enamel-, Dentin-, and Cementum-Derived Cells in Collagen-GAG Matrices In Vitro Unerupted Porcine Premolars and Molars ? Lower mandibles from 6-month old pigs. ? In aseptic environment, mandibles were split in half, soft tissue removed, and overlying bone from lingual side chiseled away. ? Exposed teeth were excised and gingiva removed. Marty-Roix R, et al., Tiss. Engr. (In press) MATERIALS AND METHODS Cell Isolation Dentin - from developing cusp tips. Mineralized enamel removed and pulp cut away. Enamel - Mineralized enamel removed from cusp tips and chiseled into small pieces. Cementum - from erupted 2nd molars and unerupted premolars. Chiseled away from tooth and into smaller pieces. Pulp removed with sterile gauze. Pulp - from base of teeth and cut into small pieces with scalpel. MATERIALS AND METHODS Methods of Cell Culture ? Cell Isolation from Digested Tissue – Tissue digested for 12 hours in collagenase. – Suspension filtered, and cells plated into tissue culture dishes. ? Explants/Cell Outgrowth – Small pieces of tissue plated onto tissue culture dishes. COLLAGEN-GAG MATRICES ? Type I (bovine tendon) ? Type II (porcine) ? Chondroitin 6-sulfate Image removed due to copyright considerations. Image removed due to copyright considerations. 1mm ? Freeze-dried ? Dehydrothermally cross-linked 500μm IV Yannas, et al. PNAS, 1989 Cell Cultures at Confluence; Digested Tissue Ameloblasts Pulp Cells Images removed due to copyright considerations. Odontoblasts Cementoblasts DENTAL TISSUES Tissue-Specific Matrix Molecules ? Enamel Amelogenin ? Dentin Dentin Matrix Protein-1 ? Cementum ? ? Pulp Cells ? Amelogenin Immunohistochemistry Amelogenin Negative ControlPre-ameloblasts Images removed due to copyright considerations. Dentin Odontoblasts Amelogenin Immunohistochemistry; Passage 1 Ameloblasts; Digested Tissue Ameloblasts; Outgrowth from Explants Images removed due to copyright considerations. Neg. Control Neg. Control 1 day Amelogenin Immunohistochemistry of Ameloblast-Seeded Collagen-GAG Matrices Images removed due to copyright considerations. 7 days Images removed due to copyright considerations. 1 day DMP-1 Immunohistochemistry of Odontoblast-Seeded Collagen-GAG Matrices 7 days 28 days SUMMARY ? Cells can be isolated from digested tissue and grown from explants: enamel, dentin , cementum, and pulp. ? Cells display distinctive characteristics. ? Ameloblasts expanded in monolayer and grown in collagen-GAG matrices express amelogenin. ? Odontoblasts in collagen-GAG matrices express DMP-1