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
• “Tissue Engineering”
– Formation of tissue in vitro for subsequent
implantation
• “Regenerative Medicine”
– Regeneration in vivo
• Coupled Approach
– Cell
Cell--seeded matrix grown in vitro to meet
certain design specifications, to facilitate
regeneration in vivo
Calcium
hydroxide
liner
Dental
Caries
Osteodentin
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
• MATRIX (SCAFFOLD)
– Porous, absorbable synthetic and natural
(collagen--GAG copolymers) biomaterials
(collagen
• CELLS (Autologous or Allogeneic)
– Differentiated cells of same type as tissue
– Stem cells (bone marrowmarrow-derived)
– Other cell types
• SOLUBLE REGULATORS
– Cytokines (growth factors) or their genes
• ENVIRONMENTAL FACTORS
– Mechanical loading
– Static and “bioreactor”
ELEMENTS FOR TISSUE
REGENERATION/ENGINEERING
CELLS
MATRIX
CYTOKINES
Autologous, Adult
(pulp and bone marrow
stromal stem cell)
Collagen--GAG
Collagen
Regulation of phenotype
Matrix biosynthesis
Page 3
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
odontoblasts),
), a wellwell-defined pulp chamber, putative
cementoblasts,, and a morphologically correct enamel.
cementoblasts
• 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): 695695-700, 2002
Growth of Porcine EnamelEnamel-, DentinDentin-, and
Cementum--Derived Cells in CollagenCementum
Collagen-GAG
Matrices In Vitro
Unerupted Porcine Premolars and Molars
• Lower mandibles from 66 -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
Marty
al.,., Tiss
Tiss.. Engr
Engr.. (In press)
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
•
•
•
•
•
•
Enamel
Dentin
Cementum
Pulp
Periodontal Ligament
Bone
500mm
WOUND HEALING
Roots of Tissue Engineering
Injury
Inflammation
(Vascularized tissue)
4 Tissue Categories
Connective Tissue
Epithelium
Nerve
Muscle
Reparative
Process
Regeneration*
CT: bone
Ep:: epidermis
Ep
Muscle: smooth
Repair (Scar)
CT: cartilage
Nerve
Muscle: cardiac, skel
skel..
*spontaneous
TISSUE ENGINEERING TRIAD
CELLS
MATRIX
CYTOKINES
Autologous, Adult
(pulp and bone marrow
stromal stem cell)
Collagen--GAG
Collagen
Regulation of phenotype
Matrix biosynthesis
Contractile actin expression
Figure removed due to copyright restrictions.
MATERIALS AND METHODS
Specimen Procurement and Tissue Dissection
Unerupted Porcine Premolars and Molars
• Lower mandibles from 66 -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.
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.
DENTAL TISSUES
Tissue -Specific Matrix Molecules
•
•
•
•
Enamel
Dentin
Cementum
Pulp Cells
Amelogenin
Dentin Matrix ProteinProtein-1
?
?
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
• 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 collagencollagen-GAG matrices express amelogenin.
• Odontoblasts in collagencollagen-GAG matrices express
DMP--1
DMP
MUSCULOSKELETAL CELLS THAT CAN EXPRESS
a -SMOOTH MUSCLE ACTIN AND CAN CONTRACT
•
•
•
•
•
•
•
•
Articular chondrocyte
Osteoblast
Meniscus fibroblast and fibrochondrocyte
Intervertebral disc fibroblast and
fibrochondrocyte
Ligament fibroblast
Tendon fibroblast
Synovial cell
M. Spector,
Mesenchymal stem cell
Wound Repair Regen
Regen..
9:11--18 (2001)
9:11
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
copyright restrictions.
50toµm
Stress
Fiber
Brock, et al., J Dent Res
2002;81:203-208
50 µm
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.
µm
5050
µm
50
µm
Brock, et al., J Dent Res
2002;81:203-208
µm
5050
µm
50
µm
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
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
• EnamelEnamel-, dentindentin-, cementumcementum-, and pulppulp- derived
cells can be grown in monolayer culture and
seeded into collagencollagen-GAG matrices.
• Enamel
Enamel-- and dentindentin- derived cells retain certain
phenotypic traits.
• Discovery that ameloblasts, odontoblasts,
cementoblasts, and pulp cells can express SMA
and can contract a collagencollagen-GAG analog of
extracellular matrix.