Essay Instructions: Outline of the dental related article with responses to the following sections:
1. Background
2. Materials and Methods
3. Results
4. Conclusions
5. Clinical Implications
The article is here below- plus I can e-mail it to you in an attachment.
TITLE: Fully functional bioengineered tooth replacement
as an organ replacement therapy
AUTHORS
Etsuko Ikedaa,b,1, Ritsuko Moritaa,c,1, Kazuhisa Nakaoa,c, Kentaro Ishidaa,c, Takashi Nakamuraa,c,
Teruko Takano-Yamamotod, Miho Ogawab, Mitsumasa Mizunoa,c,d, Shohei Kasugaie, and Takashi Tsujia,b,c,2
aDepartment of Biological Science and Technology, Faculty of Industrial Science and Technology, and cResearch Institute for Science and Technology,
Tokyo University of Science, Noda, Chiba 278-8510, Japan; bOrgan Technologies Inc., Tokyo 101-0048, Japan; dDivision of Orthodontics and Dentofacial
Orthopedics, Graduate School of Dentistry, Tohoku University, Sendai, Miyagi 980-8575, Japan; and eOral and Maxillofacial Surgery, Department
of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved June 30, 2009 (received for review March 17, 2009)
ABSTRACT:
Current approaches to the development of regenerative therapies
have been influenced by our understanding of embryonic development,
stem cell biology, and tissue engineering technology. The
ultimate goal of regenerative therapy is to develop fully functioning
bioengineered organs which work in cooperation with surrounding
tissues to replace organs that were lost or damaged as a
result of disease, injury, or aging. Here, we report a successful fully
functioning tooth replacement in an adult mouse achieved through
the transplantation of bioengineered tooth germ into the alveolar
bone in the lost tooth region. We propose this technology as a
model for future organ replacement therapies. The bioengineered
tooth, which was erupted and occluded, had the correct tooth
structure, hardness of mineralized tissues for mastication, and
response to noxious stimulations such as mechanical stress and
pain in cooperation with other oral and maxillofacial tissues. This
study represents a substantial advance and emphasizes the potential
for bioengineered organ replacement in future regenerative
therapies.
The current approaches being used to develop future regenerative
therapies are influenced by our understanding of
embryonic development, stem cell biology, and tissue engineering
technology (1??"4). One of the more attractive concepts under
consideration in regenerative therapy is stem cell transplantation
of enriched or purified tissue-derived stem cells (5), or in vitro
manipulated embryonic stem (ES) and induced pluripotent stem
(iPS) cells (6, 7). This therapy has the potential to restore the
partial loss of organ function by replacing hematopoietic stem
cells in hematopoietic malignancies (8), neural stem cells in
Parkinson’s disease (9), mesenchymal stem cells in myocardial
infarction (10), and hepatic stem cells in cases of hepatic
insufficiency (11).
The ultimate goal of regenerative therapy is to develop fully
functioning bioengineered organs that can replace lost or damaged
organs following disease, injury, or aging (4, 12??"14). The
feasibility of this concept has essentially been demonstrated by
successful organ transplantations for various injuries and diseases
(15). It is expected that bioengineering technology will be
developed for the reconstruction of fully functional organs in
vitro through the precise arrangement of several different cell
species. However, these technologies have not yet achieved
3-dimensional reconstructions of fully functioning organs. To
achieve the functional replacement of lost or damaged tissues
and organs, the development of 3-dimensional bioengineered
tissues comprising a single cell type is now being attempted using
biodegradative materials (3), appropriate cell aggregation (16),
or uniform cell sheets (17). These are now clinically applied for
corneal dysfunction (18), myocardial infarction (19), and hepatic
insufficiency (20) using oral mucosal epithelial cells, myocardial
cells, and liver cells, respectively, with favorable clinical results.
A concept has also now been proposed to develop a bioengineered
organ by reproducing the developmental processes during
organogenesis (13, 21, 22). Almost all organs arise from their
respective germs through reciprocal interactions between the
epithelium and mesenchyme in the developing embryo (23??"25).
Therefore, it is predicted that a functional bioengineered organ
could be produced by reconstituting organ germs between
epithelial and mesenchymal cells in vitro, although the existence
of organ-inductive stem cells in the adult body has not been fully
elucidated yet with the exception of hair follicles (26) and the
mammary gland (27). Tooth replacement regenerative therapy,
which is also induced by typical reciprocal epithelial, and mesenchymal
interactions (25, 28), is thought to be a feasible model
system to evaluate the future clinical application of bioengineered
organ replacement (13, 21). The strategy to develop a
bioengineered third tooth after the loss of deciduous and
permanent teeth is to properly reproduce the processes which
occur during embryonic development through the reconstitution
of a bioengineered tooth germ in vitro (21). We have recently
developed a method for creating 3-dimensional bioengineered
organ germ, which can be used as an ectodermal organ such as
the tooth or whisker follicle (29). Our analyses have provided an
effective method for reconstituting this organ germ and raised
the possibility of tooth replacement with integrated blood vessels
and nerve fibers in an adult oral environment (29). However, it
remains to be determined whether a bioengineered tooth can
achieve full functionality, including sufficient masticatory performance,
biomechanical cooperation with tissues in the oral
and maxillofacial regions, and proper responsiveness via sensory
receptors to noxious stimulations in the maxillofacial region.
There are currently no published reports describing successful
replacement with a fully functional bioengineered organ.
In our current study, we describe a fully functioning tooth
replacement achieved by transplantation of a bioengineered
tooth germ into the alveolar bone of a lost tooth region in an
adult mouse. We propose this as a model for future organ
replacement therapy. The bioengineered tooth, which was
erupted and reached occlusion in the oral environment, had the
correct tooth structure, hardness of mineralized tissues for
mastication, and responsiveness to experimental orthodontic
treatment and noxious stimulation in cooperation with tissues in
the oral and maxillofacial regions. Our results thus demonstrate
the potential of bioengineered organ replacement for use in
future regenerative therapies.
Results
Eruption and Occlusion of a Bioengineered Tooth. We first investigated
whether a bioengineered molar tooth germ, which was
reconstituted from embryonic day 14.5 (ED14.5) molar tooth
germ-derived epithelial and mesenchymal cells by our previously
developed organ germ method, could erupt and reach occlusion
with an opposing tooth in the mouse adult oral environment
(Fig. 1A). After 5??"7 days in an organ culture, a single bioengineered
molar tooth germ, which had developed at the early bell
stage of a natural tooth germ and was with a mean length of
534.4 45.6 m(Fig. 1B), was then transplanted with the correct
orientation into a properly-sized bony hole in the upper first
molar region of the alveolar bone in an 8-week-old adult murine
lost tooth transplantation model. In this model, the upper first
molar had been extracted, and the resulting wounds had been
allowed to heal for 3 weeks (Fig. 1A and Fig. S1A). The cusp tip
of the bioengineered tooth was exposed into the oral cavity at
36.7 5.5 days after transplantation at a frequency of 34/60
(56.6%) (Fig. 1C Center and Fig. S1 B??"D Center). In current
transplantation model, the non-erupted explants also occurred
at low frequency and were due to the microsurgery for the
transplantation, such as transplantation with the reverse direction
or the falling off the explants. The vertical dimension of the
tooth crown continually increased and the bioengineered tooth
finally reached the plane of occlusion with the opposing lower
first molar at 49.2 5.5 days after transplantation (Fig. 1C Right,
and Fig. S1 B??"D Right and E). During the course of eruption and
occlusion, the alveolar bone at the bony hole gradually healed in
the areas around the bioengineered tooth and the regenerated
tooth had sufficient periodontal space between itself and the
alveolar bone (Fig. 1D and Fig. S1D). The bioengineered tooth
also formed a correct structure comprising enamel, ameloblast,
dentin, odontoblast, dental pulp, alveolar bone, and blood
vessels (Fig. 1D). It is known that mice have a considerable
amount of cellular cementum that increases in thickness both on
the sides of the roots and in the interradicular area and forms
around the apex of the molar roots (30). The fully occluded
bioengineered tooth was also observed to have a large amount
of cellular cementum that was equivalent to a normal murine
molar tooth (Fig. 1D and Fig. S1A). The root of the bioengineered
tooth was also observed to be surrounded by sufficient
periodontal ligaments (PDL) (Fig. 1D). Observations of the
bioengineered tooth morphology revealed that the crown had
plural cusp structure. The lengths and crown widths of the
erupted bioengineered teeth were 1,474.4 115.1 and 690.7
177.7 m, respectively. However, the bioengineered tooth was
smaller than the other normal teeth, since at present we cannot
regulate the crown width, cusp position, and tooth patterning
including anterior/posterior and buccal/lingual structures using
in vitro cell manipulation techniques.
We also transplanted green fluorescence protein (GFP)-
labeled bioengineered tooth germ, which was reconstituted by
normal epithelial cells and the mesenchymal cells from GFPtransgenic
mice into non-transgenic mice as described above
(29). A GFP-labeled bioengineered tooth was produced and
could be observed in the bony hole in the alveolar bone of adult
mice (Fig. 1E and Fig. S1F). GFP-positive mesenchymal cells
were also detectable both in the odontoblasts and in the dental
pulp and PDL, which differentiate from the dental papilla and
dental follicle cells, respectively (Fig. 1F). Green fluorescence
was also observed in the dentinal tubules of the GFP-positive
odontoblasts in the regenerated tooth (Fig. 1F Lower).
We next investigated the gene expression profiles of colonystimulating
factor 1 (Csf1) and parathyroid hormone receptor
(Pthr1), which are thought to regulate osteoclastogenesis during pathway and at the boundary surface between the dental follicle
of the bioengineered tooth and osseous tissues, as is seen in
normal teeth (Fig. S2). These observations suggest that the
eruption of the bioengineered tooth germ faithfully reproduced
the molecular mechanisms involved in the normal tooth eruption
process.
We next analyzed the occlusion established between the
bioengineered tooth and the opposing lower teeth. We often
observed that the bioengineered tooth moved physiologically
before achieving the occlusion during the transplantation experiments.
The regenerated tooth achieved normal occlusion in
harmony with other teeth in the recipient animal and had
opposing cuspal contacts that maintained the proper occlusal
vertical dimensions between the opposing arches (Fig. 1 G and
H, and Fig. S1 B??"E). Following the achievement of occlusion at
49.2 5.5 days after transplantation, there was no excessive
increase in the tooth length or perforation of the maxillary sinus
by the erupted bioengineered tooth at up to 120 days after
transplantation. These results indicated that the bioengineered
tooth moved in response to mechanical stress and achieved
functional occlusion with the opposing natural tooth.
Masticatory Potential of the Bioengineered Tooth. The masticatory
potential of a bioengineered tooth is essential for achieving
proper tooth function (32). We thus performed a Knoop hardness
test, which is a test for mechanical hardness and is used in
particular for very brittle materials or thin sheets. This was an
important parameter for evaluating masticatory functions in our
bioengineered tooth, including both the dentin and the enamel
components. The Knoop hardness of both the enamel and dentin
of normal teeth in 3-week-old and 9-week-old mice significantly
increases in according to the postnatal period (Fig. 2). These
values for enamel and dentin in the normal teeth of 9-week-old
adult mice were measured at 447.7 88.9 and 88.4 10.2 Knoop
hardness number (KHN), respectively (Fig. 2). The same measurements
in the bioengineered tooth were 461.1 83.2 and
81.4 7.53 KHN, respectively (Fig. 2). These findings indicated
that the hardness of the bioengineered tooth is in the normal
range.
Bioengineered Tooth Response to Mechanical Stress. It has been
postulated that regeneration of a fully functional tooth could be
achieved by fulfilling critical functions in an adult oral environment
such as the cooperation of the bioengineered tooth with the
oral and maxillofacial regions through the PDL (31, 33). Histochemical
analysis of the PDL of our bioengineered tooth (Fig.
1D) showed a positive connection between this tooth and the
alveolar bone, and suggesting that this tooth may be responsive
to mechanical stress. It has been demonstrated previously that
alveolar bone remodeling is induced via the response of the PDL
to mechanical stress such as the treatment of orthodontic
movements (31, 33). These same studies have further demonstrated
that the localization of osteoclasts for bone resorption
and osteoblasts for bone formation can be observed in the area
of compression and on the tension side, respectively (31, 33).
Thus, we analyzed the movement of our bioengineered tooth and
also the osteoclast and osteoblast localization for remodeling in
the alveolar bone by inducing orthodontic movements experimentally.
When the bioengineered tooth was moved buccally for 17 days
with a mechanical force in an experimental tooth movement
model, it performed as well as a normal tooth (Fig. 3A and Fig.
S3). Histochemical analysis additionally revealed morphological
changes in the PDL in both the sides containing lingual tension
and buccal compression following 6 days of treatment (Fig. 3A
and Fig. S3). Osteoblast-like cells, which have a cuboidal
shape and rounded nuclei, and osteoclast-like cells, which are
multinucleated giant cells, were observed on the surface of the
alveolar bone within the tension and compression sides, respectively
(Fig. 3A and Fig. S3). During experimental tooth movement,
tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like giant cells were dominant on the compression side
(Fig. 3B). In contrast, the localization of osteocalcin (Ocn)
mRNA-positive cells was observed in the cells on the tension
side, indicating that osteoblast-like cells were dominant (Fig.
3B). A fluorescent double-labeling experiment using calcein and
tetracycline further showed that incorporation of these reagents
into the alveolar bone on the tension side, but not the compression
side, was clearly observable in the double-labeled line within
10 days of the orthodontic treatment (Fig. 3C). These findings
suggest that the PDL of the bioengineered tooth successfully
mediates bone remodeling via the proper localization of osteoclasts
and osteoblasts in response to mechanical stress.
Perceptive Potential of Neurons Entering the Tissue of the Bioengineered
Tooth. The perception of noxious stimulations such as
mechanical stress and pain, are important for the protection and
proper functions of teeth (34). Neurons in the trigeminal ganglion,
which innervate the pulp and PDL, can detect these stress
events and transduce the corresponding perceptions to the
central nervous system (34). We have previously reported that
nerve fibers are detectable in the pulp of a developing bioengineered
tooth in the oral cavity (29). In our current experiments,
we evaluated the responsiveness of nerve fibers in the pulp and
PDL of the bioengineered tooth to induced noxious stimulations.
Anti-neurofilament (NF)-immunoreactive nerve fibers were
detected in the pulp, dentinal tubules, and PDL of the bioengineered
tooth as in a normal tooth (Fig. 4A and Fig. S4).
Neuropeptide Y (NPY), which is synthesized in sympathetic
nerves (34), was also detected in the pulp and PDL neurons (Fig.
4A and Fig. S4 C and D). Calcitonin gene-related peptide
(CGRP), which is synthesized in sensory nerves and is involved
in sensing tooth pain (34) was also observed in both pulp and
PDL neurons (Fig. 4A and Fig. S4 E and F). NPY and CGRP
were detected in both the anti-NF positive and negativeimmunoreactive
neurons (Fig. 4A and Fig. S4 C??"F).
We next evaluated the perceptive potential of these neurons
in the bioengineered tooth against noxious stimulations such as
orthodontic treatment and pulp stimulation. The expression of
galanin, which is a neuropeptide involved in pain transmission
(35), increased in response to persistent painful stimulation of
the nerve terminals within the PDL of the bioengineered tooth
to the same extent as in a normal tooth (Fig. 4B). Thus PDL
nerve fibers in the bioengineered tooth appear to respond to
nociceptive stimulation caused by our experimental tooth movements.
Previous studies have reported that neurons expressing
the proto-oncogene c-Fos protein are detectable in the superficial
layers of the medullary dorsal horn following noxious
stimulations such as electrical, mechanical and chemical stimulation
of intraoral receptive fields involving the tooth pulp, PDL,
and peripheral nerves innervating the intraoral structures (34,
35). We found in our current analyses that the c-Fosimmunoreactive
neurons present in both the normal tooth and
the bioengineered tooth drastically increased at 2 h after experimental
tooth movement, and then gradually decreased within
48 h (Fig. 4C). Following pulp stimulation, positive neurons in
both normal and bioengineered teeth also increased at 2 h after
stimulation, but could not be detected at 48 h (Fig. 4D). These
data indicate that the nerve fibers innervating both the pulp and
PDL of the bioengineered tooth have perceptive potential for
nociceptive stimulations and can transduce these events to the
central nervous system (the medullary dorsal horn).
Discussion
We successfully demonstrate herein that our bioengineered tooth
germdevelops into a fully functioning toothwith sufficient hardness
for mastication and a functional responsiveness to mechanical stress
in the maxillofacial region. We also show that the neural fibers that
have re-entered the pulp and PDL tissues of the bioengineered
tooth have proper perceptive potential in response to noxious
stimulations such as orthodontic treatment and pulp stimulation These findings indicate that bioengineered tooth generation techniques
can contribute to the rebuilding of a fully functional tooth.
Critical issues in tooth regenerative therapy are whether the bioengineered
tooth can reconstitute functions such as mastication (32) and
responsive potential to mechanical stress (31, 33) and noxious stimulations
(34), including cooperation of the regenerated tooth with both
the oral and maxillofacial regions. Eruption and occlusion are essential
first steps toward dental organ replacement therapy and successful
incorporation into the oral and maxillofacial region (21, 36). Our
laboratory has demonstrated previously that a bioengineered tooth
germ can develop into a tooth with the correct structure in an adult
mouse(29). It has also been reported previously that normaltooth germ
isolated from murine embryos and a bioengineered tooth constructed
from cultured tooth bud cells can develop and erupt in a toothless oral
soft tissue region (diastema) of adult mice and in the tooth extraction
sockets of an adult rat (37??"42). In our current study, we provide
evidence that a bioengineered toothwith the same hardness as an adult
natural tooth can eruptwith normalgene expression, including Csf1and
Pthr1, which are thought to regulate osteoclastogenesis, and achieve
functional occlusion with the opposing natural teeth. Previous reports
have suggested that the eruption of tooth germ is generally induced at
the site of tooth development and by the gubernacular cord, which is
derived from the epithelium of the dental lamina (43). Hence, our
findings provide significant insights into tooth eruption mechanisms
and strongly suggest that masticatory potential can be successfully
restored by the transplantation of bioengineered tooth germ.
To establish cooperation between the bioengineered tooth and
the maxillofacial region, 1 critical issue to address is whether a
functional PDL is achieved and thereby the restoration of interactions
between the bioengineered tooth and the alveolar bone (31,
33). The PDL has essential roles in tooth support, homeostasis, and
repair, and is involved in the regulation of periodontal cellular
activities such as cell proliferation, apoptosis, the secretion of
extracellular matrices, the resorption and repair of the root cementum,
and remodeling of the alveolar bone proper (31, 33).Although
implant therapy has been established and is effective for replacement
of a missing tooth, this therapy involves osseointegration into
the alveolar bone that does not reconstitute the PDL (44). The
regeneration of PDL has been studied previously using cell sheets
(17) and stem cells (22), but has not yet been fully successful. It is
thought that orthodontic tooth movement, a process involving
pathogenic and physiologic responses to extreme forces applied to
a tooth through bone remodeling controlled by osteogenesis and
osteoclastgenesis (31), is a good assay model for the evaluation of
PDL functions. In our present study, the PDL associated with the
bioengineered tooth performed in complete cooperation with the
oral and maxillofacial regions and bone remodeling successfully
occurred following the application of orthodontic mechanical force.
These findings indicate that it is possible to restore and re-establish
cooperation between the bioengineered tooth and maxillofacial
regions and thus regenerate critical dental functions.
The peripheral nervous system plays important roles in the
regulation of organ functions and the perception of external
stimuli such as pain and mechanical stress (45). During development
of the peripheral nervous system, growing axons navigate
and establish connections to their developing target organs
(46). The recovery of the nervous system, which is associated
with the reentry of nerve fibers, is critical for organ replacement
(47). Although the functions of several internal organs, including
the liver, kidney, and pancreas, are also mediated by specific
humoral factors such as hormones and cytokines via blood
circulation (45), perceptions of external stimuli are also essential
to the functions of several organs, such as the eye, limbs, and
teeth (45). The tooth is well recognized as a peripheral target
organ for sensory trigeminal nerves, which are required for the
function and protection of the teeth (46). It is known also that
the perception of mechanical forces during mastication is limited
in implant patients (48). Thus, the restoration of nerve functions
is also critical for tooth regenerative therapy and future organ
replacement therapy (13, 45). In our current study, we demonstrate
that several species of nerve fibers, including NF, NPY,
CGRP, and galanin-immunoreactive neurons, successfully reentered
both the pulp and/or PDL region of the bioengineered
tooth. These nerves could thereby transduce the signals from
noxious stimulations such as mechanical stress by orthodontic
treatment and the exposure of pulp. Previous studies have also
revealed that trigeminal nerve fibers navigate and establish their
axonal projections into the pulp and PDL during early tooth
development in a spatiotemporally controlled manner through
expression of regulatory factors such as nerve growth factor, glial
cell line-derived neurotrophic factor, and semaphorin 3a (46).
Our present results suggest the possibility that the transplantation
of regenerated tooth germ can induce trigeminal axon
innervation and establishment in an adult jaw through the
replication of trigeminal axon pathfinding and nerve fiber patterning
during early tooth development (46).
In conclusion, this study provides evidence of a successful
replacement of an entire and fully functioning organ in an adult
body through the transplantation of bioengineered organ germ,
reconstituted by single cell manipulation in vitro. Our study
therefore makes a substantial contribution to the development
of bioengineering technology for future organ replacement
therapy. Further studies on the identification of available adult
tissue stem cells for the reconstitution of a bioengineered tooth
germ and the regulation of stem cell differentiation into odontogenic
cell lineage will help to achieve the realization of tooth
regenerative therapy for missing teeth.
Methods
Transplantation. The upper first molars of 5-week-old C57BL/6 (SLC) mice were
extracted under deep anesthesia. Mice were maintained for 3 weeks to allow
for natural repair of the tooth cavity and oral epithelium. Before transplantation,
we confirmed using microCT analysis that the remaining tooth root
components and/or the tooth that had developed from them could not be
observed in the bony holes (SI Methods). Following repair, an incision of
approximately 1.5 mm in length was made through the oral mucosa at the
extraction site with fine scissors to access the alveolar bone. A fine pin vice
(Tamiya) was used to create a bony hole of about 0.5??"1.0 mm in diameter in
the exposed alveolar bone surface. Just before transplantation, we removed
the collagen gel from the bioengineered tooth germ in the in vitro organ
culture and marked the top of the dental epithelium with vital staining dye,
such as methylene blue, to ensure the correct direction of the explants. The
explants were then transplanted into the bony hole according to the dye. The
incised oral mucosa was next sutured with 8??"0 nylon (8??"0 black nylon 4 mm
1/2R, Bear Medic Corp.) and the surgical site was cleaned. The mice containing
the transplants were fed a powdered diet (Oriental Yeast) and skim milk until
the regenerated tooth had erupted.
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