ch11_dentalte

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Unformatted text preview: Massachusetts Massachusetts Institute of Technology Harvard Medical School Brigham and Women’s Hospital VA Boston Healthcare System 2.782J/3.961J/BEH.451J/HST524J DENTAL TISSUE ENGINEERING M. Spector, Ph.D. Figure by MIT OCW. Page 1 REGENERATION STRATEGY REGENERATION • “Tissue Engineering” – Formation of tissue in vitro for subsequent in implantation implantation • “Regenerative Medicine” – Regeneration in vivo in • Coupled Approach – Cell-seeded matrix grown in vitro to meet Cellin certain certain design specifications, to facilitate regeneration in regeneration in vivo Calcium hydroxide liner Dental Caries Osteodentin Secondary Secondary dentin Secondary dentin Tissue Engineering Enamel Regeneration Dentin Regeneration Pulp Regeneration Restorative Materials Ceramics: porcelain Metals: amalgam, Implants Polymer: composite, gutta percha Figure by MIT OCW. After J-W. Hwang. Page 2 TISSUE ENGINEERING TISSUE • MATRIX (SCAFFOLD) – Porous, absorbable synthetic and natural (collagen(collagen-GAG copolymers) biomaterials • CELLS (Autologous or Allogeneic) – Differentiated cells of same type as tissue – Stem cells (bone marrow-derived) marrow– Other cell types • SOLUBLE REGULATORS – Cytokines (growth factors) or their genes • ENVIRONMENTAL FACTORS – Mechanical loading – Static and “bioreactor” ELEMENTS FOR TISSUE REGENERATION/ENGINEERING REGENERATION/ENGINEERING CELLS MATRIX CYTOKINES Autologous, Adult (pulp and bone marrow stromal stromal stem cell) CollagenCollagen-GAG Regulation of phenotype Matrix biosynthesis Page 3 Tissue Tissue Engineering of Complex Tooth Structures on on Biodegradable Polymer Scaffolds • Cells dissociated from porcine third molar tooth buds. buds. • Cells seeded onto PLA fiber mesh and implanted in rats rats for 20 to 30 wks. • Resulting tooth structures contained dentin (odontoblasts ), a well-defined pulp chamber, putative odontoblasts), wellcementoblasts, cementoblasts , and a morphologically correct enamel. • Results suggest the presence of epithelial and mesenchymal mesenchymal dental stem cells in porcine third molar tissues. tissues. C.S. C.S. Young, et al. J Dent Res 81(10): 695-700, 2002 Dent 695- Growth of Porcine Enamel-, Dentin-, and Enamel- DentinCementumCementum-Derived Cells in Collagen-GAG CollagenMatrices In Matrices In Vitro Unerupted Porcine Premolars and Molars • Lower mandibles from 6 -month old pigs. 6• In aseptic environment, mandibles were split in in half, soft tissue removed, and overlying bone bone from lingual side chiseled away. • Exposed teeth were excised and gingiva removed. removed. MartyMarty -Roix R, et al., Tiss . Engr. (In press) et Tiss. Engr. Page 4 COLLAGEN-GAG MATRICES • Type I (bovine tendon) • Type II (porcine) • Chondroitin 6-sulfate 1mm • Freeze -dried • Dehydrothermally cross-linked IV Yannas, et al . PNAS, 1989 DENTAL TISSUES DENTAL • • • • • • Enamel Dentin Cementum Pulp Periodontal Ligament Bone 500m m WOUND HEALING WOUND Roots of Tissue Engineering Injury Injury Inflammation (Vascularized tissue) 4 Tissue Categories Connective Tissue Epithelium Nerve Muscle Reparative Process Regeneration* CT: bone Ep: Ep: epidermis Muscle: Muscle: smooth Repair (Scar) CT: cartilage Nerve Nerve Muscle: Muscle: cardiac, skel. skel. *spontaneous TISSUE ENGINEERING TRIAD CELLS MATRIX CYTOKINES Autologous, Adult (pulp and bone marrow stromal stromal stem cell) CollagenCollagen-GAG Regulation of phenotype Matrix biosynthesis Contractile actin expression Figure removed due to copyright restrictions. MATERIALS AND METHODS MATERIALS Specimen Procurement and Tissue Dissection Unerupted Porcine Premolars and Molars • Lower mandibles from 6 -month old pigs 6• In aseptic environment, mandibles were split in in half, soft tissue removed, and overlying bone bone from lingual side chiseled away. • Exposed teeth were excised and gingiva removed. removed. MATERIALS AND METHODS MATERIALS Cell Isolation Dentin - from developing cusp tips. Mineralized enamel enamel removed and pulp cut away. Enamel - Mineralized enamel removed from cusp tips tips and chiseled into small pieces. Cementum - from erupted 2nd molars and unerupted unerupted premolars. Chiseled away from tooth and and into smaller pieces. Pulp removed with sterile sterile gauze. Pulp - from base of teeth and cut into small pieces with 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 culture dishes. • Explants/Cell Outgrowth – Small pieces of tissue plated onto tissue culture dishes. dishes. DENTAL TISSUES DENTAL Tissue -Specific Matrix Molecules • • • • Enamel Dentin Cementum Pulp Cells Amelogenin Dentin Matrix Protein-1 Protein? ? Amelogenin Immunohistochemistry Amelogenin Pre-ameloblasts Negative Control Dentin Odontoblasts Photos removed due to copyright restrictions. DMP-1 Immunohistochemistry of Odontoblast-Seeded Collagen-GAG Matrices Photos removed due to copyright restrictions. SUMMARY 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. collagen• Odontoblasts in collagen-GAG matrices express collagenDMPDMP-1 MUSCULOSKELETAL MUSCULOSKELETAL CELLS THAT CAN EXPRESS a -SMOOTH MUSCLE ACTIN AND CAN CONTRACT • • • • • • • • Articular chondrocyte Osteoblast Meniscus fibroblast and fibrochondrocyte Intervertebral disc fibroblast and fibrochondrocyte fibrochondrocyte Ligament fibroblast Tendon fibroblast Synovial cell M. Spector, Mesenchymal stem cell Wound Repair Regen. Regen. 9:119:11-18 (2001) α -Smooth Muscle Actin Content Western Blot Analysis Figure removed due to copyright restrictions. Matrix comparing smooth muscle cell controls with pulp cells. Brock, et al ., J Dent Res 2002;81:203-208 Porcine Pulp Cells in Monolayer α -Smooth Muscle Actin (red) Immunohistochemistry Photos removed due to m 50 µ copyright restrictions. Stress Fiber Brock, et al ., J Dent Res 2002;81:203-208 50 µ m Brock, et al ., J Dent Res 2002;81:203-208 Photos removed due to copyright restrictions. 0.2 mm 0.2 mm Porcine Pulp Cell-Seeded Collagen-GAG Matrices 0.2 mm 0.2 mm Contraction of Pulp Cell-Seeded Collagen-GAG Matrices Non-Seeded 10 Cell-Seeded Diameter, mm 8 6 4 2 0 0 10 20 30 Time, days Brock, et al ., J Dent Res 2002;81:203-208 H&E Pulp Cell-Seeded Collagen-GAG Matrices (28 days) SMA IH Photos removed due to copyright restrictions. µ 5050m mm µ50 µ Brock, et al ., J Dent Res 2002;81:203-208 µ 5050m mm µ50 µ SMA Immunohistochemistry SMA Immunohistochemistry Photos removed due to copyright restrictions. Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 Ameloblasts in Monolayer Culture Photos removed due to copyright restrictions. SMA Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 Odontoblasts in Monolayer Culture Photos removed due to copyright restrictions. SMA Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 d Cementoblasts in Monolayer Culture Photos removed due to copyright restrictions. SMA Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 a -Smooth Muscle Actin: Western Blot Analysis Photos removed due to copyright restrictions. Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 Contraction of Collagen-GAG Matrices by Dental Tissue Cells Cell-Mediated Contraction, % 60 50 40 30 20 Ameloblasts Cementoblasts Odontoblasts 10 0 0 5 10 15 20 25 30 Time in culture, days Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 Cell-Mediated Contraction/DNA, %/ m g Contraction of Collagen-GAG Matrices by Dental Tissue Cells 120 100 Ameloblasts Cementoblasts Odontoblasts 80 60 40 20 0 Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 1 7 14 Time in Culture, days 28 SMA Immunohistochemistry of Cell-Seeded Collagen- GAG Matrices Ameloblasts Photos removed due to copyright restrictions. Marty-Roix, et al . Tiss. Engr. 2003;9:175-186 DENTAL TISSUE CELLS WITH DENTAL MUSCLE MUSCLE • Tissue formation • Remodeling • Disease processes • Healing • Tissue engineering impart ECM architecture tooth eruption alter ECM architecture contracture wound closure contraction of scaffolds FINDINGS TO DATE • Enamel-, dentin-, cementum-, and pulp- derived Enamel- dentin- cementumpulpcells cells can be grown in monolayer culture and seeded into collagen-GAG matrices. collagen• Enamel- and dentin- derived cells retain certain Enameldentinphenotypic phenotypic traits. • Discovery that ameloblasts, odontoblasts, cementoblasts, and pulp cells can express SMA and can contract a collagen-GAG analog of collagenextracellular matrix. ...
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