International Journal of

Molecular Sciences

Review
Biomaterial-Based Approaches for Regeneration of
Periodontal Ligament and Cementum Using
3D Platforms
Chan Ho Park 1,2, *
1 Department of Dental Biomaterials, School of Dentistry, Kyungpook National University, Daegu 41940, Korea
2 Institute for Biomaterials Research and Development, Kyungpook National University, Daegu 41940, Korea




Received: 17 July 2019; Accepted: 4 September 2019; Published: 5 September 2019


Abstract: Currently, various tissue engineering strategies have been developed for multiple tissue
regeneration and integrative structure formations as well as single tissue formation in musculoskeletal
complexes. In particular, the regeneration of periodontal tissues or tooth-supportive structures is still
challenging to spatiotemporally compartmentalize PCL (poly-ε-caprolactone)-cementum constructs
with micron-scaled interfaces, integrative tissue (or cementum) formations with optimal dimensions
along the tooth-root surfaces, and specific orientations of engineered periodontal ligaments (PDLs).
Here, we discuss current advanced approaches to spatiotemporally control PDL orientations with
specific angulations and to regenerate cementum layers on the tooth-root surfaces with Sharpey’s
fiber anchorages for state-of-the-art periodontal tissue engineering.

Keywords: biomaterials; tissue engineering; regenerative medicine; periodontal ligament; cementum;

periodontal tissues

1. Introduction

1.1. The Characteristics of Periodontal Ligament and Cementum in the Periodontal Complex
The periodontal complex has hierarchically compartmentalized architectures with structural
integrations of fibrous connective tissues and mineralized tissues surrounding tooth structures [1].
As tooth-supportive structures, three different tissues are typically composed like cementum
(the mineralized layers on the tooth-root surface), periodontal ligaments (PDLs; the fibrous connective
tissues between cementum and bone surface with specific orientations to the tooth-root surfaces),
and alveolar bone (the mineral construct to sustain teeth and tooth-associated tissues with alveolar
sockets) (Figure 1) [2–4]. Of the periodontium, a major component of the function of the alveolar
bone is to appropriately position teeth and continuously remodel structures by generating mechanical
responses against compressive or tensile forces that are exerted by various external stimulations [5–7].
These mechanical transmissions of masticatory forces around teeth could be spatially generated by
integrating fibrous connective tissues or PDLs with appropriately physical tautness, so PDL anchorages
between cementum and alveolar bone surfaces within compartmentalized tissue interfaces should
be required for the functionalized periodontal complex [3,5,8]. To form the tissue integrations as
tooth-supporting constructs, Sharpey’s fibers, which are the terminal ends of principal PDL fibers,
crucially contribute to inserting and anchoring into the cementum and the mineralized layer of bone
surfaces [1,9,10]. By integrating these multiple tissues, PDL fibrous bundles can mainly provide
supportive, remodeling, sensory, and nutritive functions during mastication and occlusion [11].
In particular, oblique or perpendicular orientations of PDLs to the tooth-root surface when viewed
coronally can play pivotal roles in generating various mechanical or biological responses during
mastication and occlusion to protect teeth and bone structures [4,8,9].

Int. J. Mol. Sci. 2019, 20, 4364; doi:10.3390/ijms20184364 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 17

[11]. In particular, oblique or perpendicular orientations of PDLs to the tooth-root surface when
Int. J. Mol.coronally
viewed Sci. 2019, 20,
can4364play pivotal roles in generating various mechanical or biological responses
2 of 17

during mastication and occlusion to protect teeth and bone structures [4,8,9].

Figure 1. Illustration of tooth structure and tooth-supportive complex. Four different types of
Figure 1. Illustration of tooth structure and tooth-supportive complex. Four different types of
periodontal ligament (PDL) fibers are typically categorized between the cementum on the tooth-root
periodontal ligament (PDL) fibers are typically categorized between the cementum on the tooth-root
surface and the alveolar bone. Adapted with permission from the reference [8].
surface and the alveolar bone. Adapted with permission from the reference [8].

Figure 1 shows that integrated fibrous connective tissues (PDLs) are categorized into four typical
Figure 1 shows that integrated fibrous connective tissues (PDLs) are categorized into four typical
types: alveolar crest (radiated bundles), horizontal (perpendicular bundles), oblique (oblique bundles
types: alveolar crest (radiated bundles), horizontal (perpendicular bundles), oblique (oblique bundles
coronal-attached to bone), and apical (radiated bundles) fibers by generating optimal mechanical
coronal-attached to bone), and apical (radiated bundles) fibers by generating optimal mechanical
resistances or biological responses under various physiological loading environments [3,9]. In particular,
resistances or biological responses under various physiological loading environments [3,9]. In
the oblique PDL group, which spatially constitutes approximately 70% of the complex, mainly resists
particular, the oblique PDL group, which spatially constitutes approximately 70% of the complex,
vertical and intrusive forces during mastication applications, while other typical PDL groups generate
mainly resists vertical and intrusive forces during mastication applications, while other typical PDL
systemic responses to cope with various types of masticatory forces [9,11].
groups generate systemic responses to cope with various types of masticatory forces [9,11].
Cementum is the heterogenic mineralized layer, which is deposited on the tooth dentin surface
Cementum is the heterogenic mineralized layer, which is deposited on the tooth dentin surface
with a 50–300 µm thickness [12–14], and the biochemical compositions of cementum are similar to
with a 50–300 μm thickness [12–14], and the biochemical compositions of cementum are similar to
bone tissues with approximately 50% mineral and 50% organic components [15,16]. From the aspect
bone tissues with approximately 50% mineral and 50% organic components [15,16]. From the aspect
of tissue functions, cementum plays the crucial role of anchoring PDL fibers onto tooth-root surfaces
of tissue functions, cementum plays the crucial role of anchoring PDL fibers onto tooth-root surfaces
with Sharpey’s fibers, which are critical for periodontal functioning restorations [12,14]. Cementum is
with Sharpey’s fibers, which are critical for periodontal functioning restorations [12,14]. Cementum
generally classified into two different types of acellular and cellular cementum tissues, according to
is generally classified into two different types of acellular and cellular cementum tissues, according
the location and function of human teeth [13,14]. Briefly, the acellular cementum contributes to the
to the location and function of human teeth [13,14]. Briefly, the acellular cementum contributes to the
tooth attachment of principal fibers of PDLs for periodontal functions, while cellular cementum is
tooth attachment of principal fibers of PDLs for periodontal functions, while cellular cementum is
deposited around the apical regions of tooth roots for the positional adaptations of teeth to occlusion
deposited around the apical regions of tooth roots for the positional adaptations of teeth to occlusion
for periodontal repair [1,14,17].
for periodontal repair [1,14,17].
Due to the tissue’s significance, such as the morphological and functional characteristics
Due to the tissue’s significance, such as the morphological and functional characteristics of
of cementum, various studies have been undertaken to understand and to attempt to develop
cementum, various studies have been undertaken to understand and to attempt to develop
cementum regeneration approaches for periodontal tissue integrations and functional PDLs [11,13,18].
cementum regeneration approaches for periodontal tissue integrations and functional PDLs
Although cementogenesis is anticipated to lead to the promotion of a new fibrous tissue attachment
[11,13,18]. Although cementogenesis is anticipated to lead to the promotion of a new fibrous tissue
process for periodontal tissue engineering, it still remains a major challenge to regulate the optimal
attachment process for periodontal tissue engineering, it still remains a major challenge to regulate
dimension formations without ankylosis, the bone fusion to the tooth-root surfaces, and to develop the
the optimal dimension formations without ankylosis, the bone fusion to the tooth-root surfaces, and
highly-predictable techniques for cementogenic procedure for PDL attachments [1,12,14].
to develop the highly-predictable techniques for cementogenic procedure for PDL attachments
[1,12,14].
1.2. Periodontal Disease and Treatments
Periodontal
1.2. Periodontal disease
Disease andor periodontitis, which is a common inflammatory chronic disease, causes
Treatments
irreversible periodontal complex destruction, disconnection of principal fibrous ligamentous tissues,
and consequent loss of barrier function [19–21]. The invasion of oral bacteria or the formation of
dental biofilm (or the maturation of dental plaque) on tooth surfaces are mainly associated with
Int. J. Mol. Sci. 2019, 20, 4364 3 of 17

pathogenesis of periodontal disease caused by host inflammatory responses [21,22]. In addition to

periodontal pathogens, environmental and host genetic factors could also disturb severe periodontal
manifestations to induce periodontitis [23]. In general, non-surgical periodontal treatments have been
widely performed, such as scaling or root planing to mechanically remove periodontal pathogens,
or anti-infective treatments to biochemically eliminate bacteria or microbial biofilm [24]. In the case of
surgical treatments, the guided tissue regeneration (GTR)/guided bone regeneration (GBR) techniques
are commonly employed for periodontal complex regenerations with osteoconductive/osteoinductive
materials or bioactive biologics [5,25–28]. However, various therapeutic strategies for periodontal
tissue regenerations are still too unpredictable and uncontrollable to form multiple tissue formation for
fiber-mineral tissue complexes, spatial tissue compartmentalization with micron-scaled dimensions,
specific angulation structures of PDLs, and tissue integrations for functioning restorations as
tooth-supportive structures [5,24,29]. Therefore, severe cases of periodontitis around natural tooth
structures could result in loosening teeth and would still be required after natural tooth extractions for
dental prosthetics.
Recently, regenerative strategies have been investigated for pre-clinical and clinical situations
and developed for periodontal tissues, like implantable scaffolds or biologic delivery systems [30–32].
Major approaches in periodontal tissue engineering have focused on the development of bone
substitutes or alveolar bone regeneration materials for dental prosthetic stabilities or natural tooth
preservations [33,34]. However, the regeneration and configuration of PDL-cementum complexes still
depends on biomechanical optimization during mastication or existing stem cell-like cell activations,
like proliferation or differentiation in PDL-cementum interfaces for periodontal revitalization, following
histophysiological adaptations of the periosteum [35,36]. In particular, due to the difficulties in 1)
reconstituting 3D structures of PDLs and cementum along the tooth surfaces, 2) controlling specific
orientations of fibrous connective tissues, and 3) guiding hierarchical multiple tissue formations within
approximately 500 µm interfaces, it has remained challenging [18,24,37]. The described limitations
cause unpredictable and unsatisfactory regenerations of tooth-supportive complex formations by
the attachments of both mineralized and ligamentous tissues, as well as functional restorations of
neogenic periodontal tissues [24,38]. In this review, recent advances are introduced with the individual
3D platforms for angularly engineered PDLs and the specific biomaterial fabrication for cementum
regeneration with Sharpey’s fiber insertions.

2. PDL Regenerations with Angular Organization Using Topographic Approaches of 3D

Platforms
Although multilayered scaffolds [32,39,40] and PDL cell sheet technology [2,41,42] have facilitated
spatially compartmentalized hierarchical tooth-supportive structures with specific dimensions for
individual periodontal tissues, it is challenging for fibrous tissues to be directionally controlled using
micron-scaled, engineered architecture. In particular, topological strategies on 2D substrates have
been actively developed to control the angular organizations of PDL cells [43–45] as well as other
fibrous connective tissue cells [46,47]. Takahashi et al. fabricated a thermoresponsive brush surface
using poly(N-isopropylacrylamide) (PIPAAm) to obtain selective patterns and regulate human dermal
fibroblast alignments [47]. The results demonstrated that cultured fibroblasts on chemically patterned
surfaces showed increased cell populations while maintaining the orientation for five days in vitro [47].
Moreover, engineered cell sheets could be detached from the fabricated culture dishes with the angular
orientations and easily transplanted to target tissues or organs, which require the specific angulations
of tissues [47].
Although there have been many efforts to control and regulate the orientations of fibrous
connective tissue cells based on the developments of substrate topologies, they are significantly
limited to investigate 3D platforms for the perpendicular/oblique orientations of PDLs to tooth-root
surfaces. Recently, several techniques have been developed to physically control the orientations or
angulations of PDLs using the fiber-guidable 3D microstructures of scaffolds, which could be created by
Int. J. Mol. Sci. 2019, 20, 4364 4 of 17
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 17

surfaces. Recently, several techniques have been developed to physically control the orientations or
directional freeze-casting,
angulations of PDLs usingadditive manufacturing,
the fiber-guidable or soft lithographic
3D microstructures methods
of scaffolds, which[3]. Thebe
could freeze-casting
created
method with temperature gradients could simply create longitudinal pore architectures
by directional freeze-casting, additive manufacturing, or soft lithographic methods [3]. The byfreeze-
ice crystal
formation
castingand control
method thetemperature
with directionalgradients
internal architectures in gelatin
could simply create hydrogels
longitudinal porewith predictability
architectures by [3].
In particular,
ice crystaliceformation
crystal growth kinetics
and control theby different internal
directional freezingarchitectures
mold surfaces spatially
in gelatin created structural
hydrogels with
predictability
similarities [3]. InPDLs,
to natural particular,
3–10 ice
µmcrystal
diameter growth kinetics by different freezing
of cementum-associated and 10–20mold µm surfaces
diameter
spatially created structural similarities to natural PDLs, 3–10 μm diameter of
of bone-associated PDL bundles [3]. After removing ice crystals from frozen gelatin scaffolds by cementum-associated
and 10–20 μm
freeze-drying, diameter of longitudinal
directionally bone-associated PDLstructures
pore bundles [3]. After have
could removing ice crystals
oblique from frozen
orientations or radial
gelatin scaffolds by freeze-drying, directionally longitudinal pore structures could have oblique
angulations to the tooth-root surfaces (Figure 2). The study demonstrated that the freeze-casting method
orientations or radial angulations to the tooth-root surfaces (Figure 2). The study demonstrated that
could create oblique PDL structures, which constitute approximately 70% of the PDL architectures,
the freeze-casting method could create oblique PDL structures, which constitute approximately 70%
even of
though it isarchitectures,
the PDL difficult to make other PDL
even though it is structures
difficult to with
makeradial
other and
PDL horizontal orientations.
structures with radial andIn the
results, the freeze-casted
horizontal orientations.hydrogels can be
In the results, thethe 3D platform
freeze-casted in ordercan
hydrogels to be
guide
the 3Dangularly
platformorganized
in order toPDLs
with guide
anatomically
angularly structural
organizedsimilarities
PDLs with to natural oblique
anatomically PDLsimilarities
structural bundles [3]. to natural oblique PDL
bundles [3].

Figure 2. Freeze-casting
Figure 2. Freeze-castingmethod
method for for longitudinal porestructures
longitudinal pore structures with
with controlled
controlled angulations.
angulations.
Depending on freezing
Depending directions
on freezing directions(the
(thefirst
first row), variousangular
row), various angularorganizations
organizations of internal
of internal pore pore
architectures were created and analyzed using micro-computed tomographic images with both
digitized 2D cross-sectional and 3D reconstructed images (the second row), 3D surface mapping to
show angulations to the surface of the paraffin tooth (the third row), and scanning electron microscopic
(SEM) images for morphologies (the fourth row). Adapted with permission from the reference [3].
 architectures were created and analyzed using micro-computed tomographic images with both
digitized 2D cross-sectional and 3D reconstructed images (the second row), 3D surface mapping to
show angulations to the surface of the paraffin tooth (the third row), and scanning electron
microscopic
Int. J. Mol. (SEM)
Sci. 2019, 20, 4364 images for morphologies (the fourth row). Adapted with permission from the5 of 17
reference [3].

The
The additive
additive manufacturing
manufacturing or or 3D3D printing
printing technique
technique has has been
been recently
recently addressed
addressed to to create
create
micro-groove patterns on PDL scaffold surfaces with specific pattern intervals
micro-groove patterns on PDL scaffold surfaces with specific pattern intervals [8,32,37]. In general, [8,32,37]. In general,
the
the additive
additive manufacturing
manufacturing technique
technique can can generate
generate groove
groove patterns
patterns onon the
thefeatures.
features. Patterns
Patterns areare
considered as topographical artifacts (or so-called stair-stepping errors), which
considered as topographical artifacts (or so-called stair-stepping errors), which should be removed should be removed
to
to obtain
obtain smooth
smooth surfaces.
surfaces. Park
Park et et al.
al. reinterpreted
reinterpreted the the artifactual
artifactual patterns
patterns asas micro-groove
micro-groove patterns
patterns
with
with high
highpredictability
predictability and and developed
developed three three different
differentangular
angularpatterns
patterns with
withoptimal
optimal micro-groove
micro-groove
intervals
intervals for
for human
human PDL PDL cellcell alignments
alignments in inmanufacturing
manufacturing procedures.
procedures. Additive
Additive layer
layer thickness,
thickness,
which
whichisisreflected
reflectedin micro-groove
in micro-groove intervals, can becan
intervals, configured for the surface
be configured for theroughness and modeling
surface roughness and
speeds.
modeling To speeds.
optimizeTo intervals
optimize to intervals
regulate cell alignments,
to regulate cell the study designed
alignments, twodesigned
the study types of micro-groove
two types of
intervals (12.70intervals
micro-groove µm and(12.70 25.40 μm µm andintervals
25.40 between
μm intervals groove patterns)
between withpatterns)
groove in vitro with
experiments.
in vitro
The results showed
experiments. that showed
The results intervalsthat were significantly
intervals affected foraffected
were significantly angularforcell alignments
angular and cell
cell alignments
nucleus
and celldeformation, regardlessregardless
nucleus deformation, of micro-groove patterns on
of micro-groove the surfaces
patterns on theofsurfaces
scaffoldsofinscaffolds
seven-day in
cultures (Figure 3). That is, 12.70 µm micro-groove intervals showed
seven-day cultures (Figure 3). That is, 12.70 μm micro-groove intervals showed a random a random organization of cells,
while 25.40 µm
organization ofintervals
cells, whilesignificantly
25.40 μm aligned
intervalscells with parallel
significantly angulations
aligned to parallel
cells with the reference direction
angulations to
for seven days (Figure 3) [8].
the reference direction for seven days (Figure 3) [8].

Figure 3. Cell orientation controls using different micro-groove intervals with 12.70 µm and 25.40 µm
distance
Figure 3.between grooves. controls
Cell orientation After culturing human PDL
using different cells for seven
micro-groove days, with
intervals (a–c) 12.70 µm
μm micro-groove
and 25.40 μm
intervals
distance randomly organized
between grooves. cellsculturing
After but (d–f)human
25.40 µmPDLinterval substrate
cells for guided
seven days, highly
(a–c) aligned
12.70 cells.
μm micro-
Adapted with permission
groove intervals randomlyfrom the reference
organized [8].
cells but (d–f) 25.40 μm interval substrate guided highly aligned
cells. Adapted with permission from the reference [8].
Based on the interesting results of micro-groove intervals, PDL-guiding scaffolds were
manufactured
Based onwith thethree different results
interesting angulatedof micro-groove
micro-groovepatterns at 0◦PDL-guiding
intervals, , 45◦ , and 90◦ , which could
scaffolds be
were
parallel, oblique,
manufactured andthree
with perpendicular to the reference
different angulated directions,
micro-groove respectively
patterns at 0°, 45°,(Figure 4) [8].
and 90°, After
which the
could
seeding and oblique,
be parallel, in vitro culturing of human to
and perpendicular PDLthecells withindirections,
reference scaffolds, respectively
different topographical-guiding
(Figure 4) [8]. After
microarchitectures
the seeding and inon the culturing
vitro surfaces of scaffolds
human PDL could guide
cells the scaffolds,
within orientations of cells
different with angles
topographical-
(0 ◦ ◦
, 45 , and ◦
90 ) in seven days and cell angulations cultured for 21 the
days were maintained
guiding microarchitectures on the surfaces of scaffolds could guide orientations of cells with
with
higher cell populations (Figure 4). Therefore, the layer thickness configured at the
angles (0°, 45°, and 90°) in seven days and cell angulations cultured for 21 days were maintained with digital slicing
and
higherthecell
manufacturing
populations steps
(Figurecould be the critical
4). Therefore, factorthickness
the layer to determine ligamentous
configured cell orientations,
at the digital slicing and
which angulated micro-groove patterns could regulate using the 3D printing system.
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 17

Int. J.manufacturing
the Mol. Sci. 2019, 20, steps
4364 could be the critical factor to determine ligamentous cell orientations, which
6 of 17

angulated micro-groove patterns could regulate using the 3D printing system.

Figure 4. The scanning electron microscopic (SEM) images of micro-groove patterns on the surface
Figure 4. The scanning electron microscopic (SEM) images of micro-groove patterns on the surface of
of PDL architectures and fluorescence images of cell alignments in 7- and 21-day in vitro cultures
PDL architectures and fluorescence images of cell alignments in 7- and 21-day in vitro cultures for
for qualitative assessments. Three different micro-groove patterns facilitated angular organization of
qualitative assessments. Three different micro-groove patterns facilitated angular organization of
human PDL cells in 7- and 21-day cultivations. In particular, the micro-grooved topographies with
human PDL cells in 7- and 21-day cultivations. In particular, the micro-grooved topographies with
25.40 µm interval showed higher populations of cells as well as maintaining specific cell orientations.
25.40 μm interval showed higher populations of cells as well as maintaining specific cell orientations.
Adapted with permission from the reference [8].
Adapted with permission from the reference [8].
In addition to 3D manufacturing techniques and architectures for PDL orientations, PDL-guidable
In addition 2D
micro-patterned to 3DPCLmanufacturing techniques
films were fabricated andsoft
by the architectures
lithographic fortechnique,
PDL orientations, PDL-
which created
guidable micro-patterned
submicron-scaled 2D PCL
topographies onfilms
PCL werepillarsfabricated
for angular by PDL
the soft lithographic[48,49].
organizations technique,This which
study
created
investigated the optimization of the width and depths of micro-groove patterns to controlThis
submicron-scaled topographies on PCL pillars for angular PDL organizations [48,49]. the
study investigated
orientations the optimization
and thickness of the PDL
of regenerated widthfibrous
and depths of micro-groove
bundles in vitro and inpatterns
vivo [48].to The
control the
results
orientations
demonstrated andthatthickness
alignments of regenerated
of human PDL PDL fibrous
cells bundles in vitro
with newly-formed and in vivo
collagenous [48]. orientations
bundle The results
demonstrated that alignments of human PDL cells with newly-formed
corresponded with topographical characteristics by the lithographic design and manufacture, but collagenous bundle
large
orientations corresponded
inter-pillar spaces like 400 µm withdid topographical characteristics
not allow the formation by the
of oriented lithographic
fibrous tissue or design
developand the
manufacture, but large inter-pillar spaces like 400 μm did not allow the formation
density of collagen bundles (such as fiber-structure thickness) [48]. Therefore, the study demonstrated of oriented fibrous
tissue
that theor optimal
develop dimensions
the density of and collagen
geometriesbundles (such as fiber-structure
of PDL-guiding scaffolds were thickness) [48].required
critically Therefore,
for
the study demonstrated that the optimal dimensions and geometries of PDL-guiding
angular PDL and collagenous fiber bundle formations (Figure 5) [48]. As the results of the subcutaneous scaffolds were
critically required
transplantation modelfor showed,
angular the PDL and collagenous
designed dimensions fiber bundle
of the widthformations
and the depth (Figure
of the5) [48]. As the
micro-grooves
results of the subcutaneous transplantation model showed, the designed
significantly affected cell alignment and collagen bundle organization between the bone scaffold dimensions of theand
width
the
and the depth of the micro-grooves significantly affected cell alignment
dentin segment surface (Figure 5) [48]. In addition, the chemical vapor deposition (CVD) technique was and collagen bundle
organization between
applied to fabricate thethe bone of
surfaces scaffold and the dentin
polymer-based segment
PDL-guiding surface
films with (Figure 5) [48]. which
growth factors, In addition,
could
the chemical vapor deposition (CVD) technique was applied to fabricate
promote tissue regeneration within compartments for different periodontal tissues [49]. The growth the surfaces of polymer-
based PDL-guiding
factor-associated films with
periodontal growth
scaffolds factors,
could which could
synergistically promote
enhance tissue regeneration
periodontal within
tissue regenerations
compartments for different periodontal tissues [49]. The growth factor-associated
like bone, PDL, and limited cementum around exposed tooth-root surfaces in the in vivo periodontal periodontal
scaffolds
fenestrationcould synergistically
defects enhance
of the rat models periodontal tissue regenerations like bone, PDL, and limited
[49].
 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 17

Int. J. Mol. Sci. 2019, 20, 4364 7 of 17

cementum around exposed tooth-root surfaces in the in vivo periodontal fenestration defects of the
rat models [49].

Figure5.5. The
Figure The surface
surface characterizations
characterizations of of designed
designed andand manufactured
manufacturedmicro-patterned
micro-patternedpoly-ε-poly-ε-
caprolactone (PCL) films using scanning electron microscopy (SEM) and histological
caprolactone (PCL) films using scanning electron microscopy (SEM) and histological analyses of analyses of fibrous
tissue formation
fibrous following following
tissue formation different patterns.
differentThe morphological
patterns. significancesignificance
The morphological of designedofarchitectures
designed
was qualitatively
architectures wasanalyzed usinganalyzed
qualitatively SEM following
using the
SEMdesign legends
following the (first
design and second(first
legends columns). For the
and second
histologies
columns). of ForPDL-guiding
the histologiesPCL of films, hematoxylin
PDL-guiding and eosin
PCL films, (H&E) staining
hematoxylin and eosin(third
(H&E)column), Masson’s
staining (third
trichrome
column), staining
Masson’s (fourth column),
trichrome and fluorescence
staining (fourth column),staining
andusing 40 ,6-diamidino-2-phenylindole
fluorescence staining using 4′,6-
(DAPI; blue) and tubulin (green)
diamidino-2-phenylindole (DAPI; staining (fifthtubulin
blue) and column) showed
(green) fibrous(fifth
staining tissue formation
column) with fibrous
showed collagen
and cell alignments at six weeks. (D: dentin; B: bone). Adapted with permission from
tissue formation with collagen and cell alignments at six weeks. (D: dentin; B: bone). Adapted with the reference [48].
permission from the reference [48].
In addition to the soft lithographic strategy, the electrospinning fabrication technique has been
widelyInutilized
addition totomanufacture random or
the soft lithographic highlythe
strategy, aligned nano-/micro-fibrous
electrospinning fabricationmembranes
technique has in dental
been
tissue
widelyengineering
utilized toapplications
manufacturesuch as periodontal
random regeneration
or highly aligned [50–52] or pulp
nano-/micro-fibrous regeneration
membranes [53–55].
in dental
Intissue
particular, the electrospun
engineering applications2Dsuch
membranes with unidirectional
as periodontal regeneration alignments
[50–52] or pulpor controllable-aligned
regeneration [53–
patterns of nano-fibers
55]. In particular, were investigated
the electrospun for specific
2D membranes withcell orientationsalignments
unidirectional and migrations to regulate
or controllable-
cellular
alignedresponses
patterns of to designed
nano-fibers microenvironments
were investigated [56,57]. However,
for specific there is theand
cell orientations limitation to create
migrations to
3D directional
regulate internal
cellular architectures
responses to regulate
to designed cell or tissue orientations
microenvironments with structural
[56,57]. However, there is thesimilarities
limitationto
to create
PDLs 3D directional
because periodontalinternal
connectivearchitectures to regulate
tissues have cell or
the spatially tissue orientations
perpendicular with angulations
or oblique structural
tosimilarities
tooth-root to PDLs because
surfaces periodontal
as described connective
above [51,58]. Jiangtissues
et al. have the developed
recently spatially perpendicular or
3D architectures,
oblique angulations to tooth-root surfaces as described above [51,58].
which were stacked using electrospun nano-fibrous membranes and assembled with crosslinkedJiang et al. recently developed
3D architectures,
chitosan hydrogelswhich (Figurewere
6a) stacked using
[51]. After theelectrospun nano-fibrousmaterial
blended biopolymeric membranes and assembled
(poly-ε-carpolactone
with
and crosslinked chitosan
polyethylene hydrogels
glycol; PCE) (Figure 6a) [51].
was electrospun After the blended
for nano-fibrous biopolymeric
membranes, material
the PCE (poly-
membranes
were stacked layer-by-layer and assembled in chitosan solution with the genipin crosslinking PCE
ε-carpolactone and polyethylene glycol; PCE) was electrospun for nano-fibrous membranes, the agent
membranes
(Figure 6a). Inwere stackedthe
the results, layer-by-layer
cells seeded and into assembled
scaffolds were in chitosan solution
directionally alignedwithwith
the polarized
genipin
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 17
Int. J. Mol. Sci. 2019, 20, 4364 8 of 17
crosslinking agent (Figure 6a). In the results, the cells seeded into scaffolds were directionally aligned
with polarized morphologies in vitro (Figure 6b). PDL-like fibrous connective tissues were angularly
morphologies in vitro (Figure 6b). PDL-like fibrous connective tissues were angularly reorganized
reorganized perpendicularly or obliquely, as well as cell infiltration, into the 3D scaffolds in in vivo
perpendicularly or obliquely, as well as cell infiltration, into the 3D scaffolds in in vivo (Figure 6c) [51].
(Figure 6c) [51].

Theelectrospun
Figure6.6.The
Figure electrospunnano-/micro-fibrous
nano-/micro-fibrous3D 3Dscaffolds
scaffolds for
forengineered
engineered periodontal
periodontal ligament
ligament (PDL)
orientations. (a)
orientations. (a)TheTheschematic illustration
schematic shows shows
illustration 3D stacked
3D scaffold
stackedcreation
scaffold using the electrospinning
creation using the
fabrication, and chitosan solution was facilitated for 3D structures with the genipin
electrospinning fabrication, and chitosan solution was facilitated for 3D structures with the genipin crosslinking agent.
For in vivo transplantation,
crosslinking agent. For in vivo thetransplantation,
periodontal defects the around the mesial
periodontal defects root of thethe
around firstmesial
molarroot
toothofwere
the
surgically
first created
molar tooth werein asurgically
rat model. (b) Ininvitro,
created a rat the seeded
model. cells
(b) In showed
vitro, polarized
the seeded cytoskeletons
cells showed and
polarized
morphologicaland
cytoskeletons alignments using scanning
morphological alignmentselectron
usingmicroscope
scanning(SEM) electronand microscope
fluorescence(SEM)microscope
and
images (Blue: DAPI and Green: actin). The arrowed yellow-dash line
fluorescence microscope images (Blue: DAPI and Green: actin). The arrowed yellow-dash lineindicates directions of aligned
cells. (c) directions
indicates By the hematoxylin
of alignedand eosin
cells. (H&E)
(c) By staining method,
the hematoxylin PDL-like
and eosin (H&E) fibrous connective
staining method,tissues
PDL-
werefibrous
like formedconnective
with the perpendicular
tissues were orientations
formed withtothe theperpendicular
tooth-root surface in two months.
orientations to the The yellow
tooth-root
triangles
surface inindicate the limited
two months. but newly
The yellow formed
triangles cementum-like
indicate the limitedtissue
butlayer
newly andformed
the arrowed back-dash
cementum-like
lines indicate the orientations of fibrous tissues. (Bn: Bone). Adapted
tissue layer and the arrowed back-dash lines indicate the orientations of fibrous tissues. (Bn:with permission from the
Bone).
reference [51].
Adapted with permission from the reference [51].
3. Cementum Regeneration on the Tooth-Root Surface Using Biomaterial-Based 3D Engineered
3.Environments
Cementum Regeneration on the Tooth-Root Surface Using Biomaterial-Based 3D Engineered
Environments
Cementogenesis, or cementum regeneration, is a critical procedure for the new attachment process
and Cementogenesis,
structural integrationor cementum
of multipleregeneration, is a critical
periodontal tissues procedure
to generate for the new functioning
tooth-supportive attachment
process andlike
restoration structural integration
the anchorage of multiple
of collagenous periodontal
fibrous tissues
tissues, PDLs to generate
[5,12,14,37]. tooth-supportive
Various biomaterials
functioning restoration like the anchorage of collagenous fibrous tissues, PDLs
have been investigated to induce the interfacial tissue formation between the tooth dentin [5,12,14,37]. Various
and
biomaterials have been investigated to induce the interfacial tissue formation
PDL fibrous tissues using 3D biopolymeric scaffolds [59]. The poly(lactic-co-glycolic acid) (PLGA)between the tooth
dentin andplayed
scaffolds PDL fibrous tissues
a key role using 3Dcementoblastic
in carrying biopolymeric scaffolds
cells and [59]. The poly(lactic-co-glycolic
cementogenesis-promoting acid)
biologics,
(PLGA) scaffolds played a key role in carrying cementoblastic cells and cementogenesis-promoting
like platelet-derived growth factor-BB (PDGF-BB), for cementoblastic cell activation and cementogenesis
biologics,
in in vivo like platelet-derived
environments [60,61].growth
However, factor-BB (PDGF-BB), in
most investigations forcementogenesis
cementoblastichavecell activation and
mainly focused
cementogenesis in in vivo environments [60,61]. However, most investigations in
on the developments of biopolymeric carriers for delivering bioactive molecules [60,62–66], as well cementogenesis
have mainly focused on the developments
as physiological/pathological adaptationsofand biopolymeric
cementogenic carriers for delivering
differentiations ofbioactive molecules
dental stem cells in
[60,62–66],
micro-niches as [20,67–69],
well as physiological/pathological
rather than geometric oradaptations
architecturaland cementogenic
regulations differentiations
for the cementogenesis.of
dental stem cells in micro-niches [20,67–69], rather than geometric or architectural
It could be difficult for the cementum regeneration to spatiotemporally control mineralized layer regulations for the
Int. J. Mol. Sci. 2019, 20, 4364 9 of 17

depositions on the tooth-root surfaces in micron-scaled thickness, so it still depends on the biological
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 17
activations of transplanted stem cells or biologic-activated host cells/tissues.
Recently, the different
cementogenesis. It couldtopologies orfor
be difficult thethebiodegradability
cementum regenerationof biomaterials have been
to spatiotemporally investigated
control
to activate the canonical Wnt signaling or Wnt/β-catenin signaling pathway, which
mineralized layer depositions on the tooth-root surfaces in micron-scaled thickness, so it still depends is involved in
inducing cementogenesis
on the biological activationsand osteogenesis
of transplanted in thecells
stem periodontal tissue development
or biologic-activated or regeneration.
host cells/tissues.
Recently, the different
Mao et al. demonstrated topologies
that the or thematerials
bioceramic biodegradability of biomaterials
fabricated have been investigated could
to create nano-/microstructures
highly promote cementogenic differentiation using human PDL cells [70],which
to activate the canonical Wnt signaling or Wnt/β-catenin signaling pathway, whichiscould
involved in
differentiate
inducing cementogenesis and osteogenesis in the periodontal tissue development or regeneration.
cementoblast-like cells and form a hierarchically compartmentalized structure like cementum-PDL
Mao et al. demonstrated that the bioceramic materials fabricated to create nano-/microstructures
constructs
could[71–73].
highly promoteWhile traditional
cementogeniccalcium phosphate
differentiation (CaP)-based
using human bioceramic
PDL cells [70], which materials
could have
good osteoconductivity to induce bone formation, the lack of osteoinductivity
differentiate cementoblast-like cells and form a hierarchically compartmentalized structure like is required to
improve cementum formation
cementum-PDL for which
constructs [71–73]. Whilestem cell-like
traditional cellsphosphate
calcium or various biologics
(CaP)-based are commonly
bioceramic
materials
contributed have Therefore,
[74,75]. good osteoconductivity to induce bone
nano-/microstructures formation,
with the lack of specificity
3D topographic osteoinductivity
wereisrecently
required to improve cementum formation for which stem cell-like cells or various biologics are
developed to promote the cementogenic differentiation of human PDL stem cells as well as cell
commonly contributed [74,75]. Therefore, nano-/microstructures with 3D topographic specificity
attachment on the fabricated surface (Figure 7) [70]. In particular, the canonical Wnt signaling pathway,
were recently developed to promote the cementogenic differentiation of human PDL stem cells as
which is involved in the physiological
well as cell attachment process
on the fabricated of mineralized
surface (Figure 7) [70].tissue development
In particular, [76], was
the canonical Wnt activated
on the nano-/microstructured
signaling pathway, which is surfaces
involvedininorder to enhance
the physiological the cementogenic
process differentiation
of mineralized tissue development of human
PDL stem[76],cells
was [70].
activated on the nano-/microstructured surfaces in order to enhance the cementogenic
differentiation of human PDL stem cells [70].

Figure Figure
7. The 7. morphological
The morphological characterizations of
characterizations ofthe
thetraditional hydroxyapatite
traditional and the
hydroxyapatite andnano-
the nano-/
/microstructured hydroxyapatite with cementogenic expression levels in vitro. (a,c) The traditional
microstructured hydroxyapatite with cementogenic expression levels in vitro. (a,c) The traditional
hydroxyapatite bioceramic material surfaces were characterized by a scanning electron microscope
hydroxyapatite bioceramic material surfaces were characterized by a scanning electron microscope
(SEM) before and after the human PDL stem cell seeding. (b,d) The nano-/microstructure
(SEM) before and after
characterized the humanmaterials
hydroxyapatite PDL stemwerecell seeding.
evaluated (b,d)
using The
SEM nano-/microstructure
before and after human PDLcharacterized
stem
hydroxyapatite materials
cell seeding. (e,f) The were evaluated
cementogenic using levels
expression SEM before and after human
were quantitatively assessedPDL
with stem cell seeding.
cementum
attachment
(e,f) The protein expression
cementogenic (CAP) and cementum protein
levels were (CEMP). The fabricated
quantitatively assessed hydroxyapatite
with cementum surfaces
attachment
protein (CAP) and cementum protein (CEMP). The fabricated hydroxyapatite surfaces (red columns)
showed statistically significant differences from the traditional (blue columns). (* p < 0.05). Adapted
with permission from the reference [70].
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 10 of 17

Int. J. Mol. Sci. 2019, 20, 4364

(red columns) showed statistically significant differences from the traditional (blue columns). (* p 10
< of 17
0.05). Adapted with permission from the reference [70].
Although the canonical Wnt signaling pathway has been known to significantly contribute to
Although the canonical Wnt signaling pathway has been known to significantly contribute to
the tooth morphogenesis at the tooth development stage and the activation of the cementogenic
the tooth morphogenesis at the tooth development stage and the activation of the cementogenic
differentiation of PDL cells [77–81], cementoblast cells in fibrin hydrogel matrices simultaneously
differentiation of PDL cells [77–81], cementoblast cells in fibrin hydrogel matrices simultaneously
increase plasminogen expression, which promotes matrix degradation and cell apoptosis in fibrin
increase plasminogen expression, which promotes matrix degradation and cell apoptosis in fibrin
hydrogel (Figure 8) [79]. Rahman et al. investigated interactive fibrinolysis with the biomineralization
hydrogel (Figure 8) [79]. Rahman et al. investigated interactive fibrinolysis with the biomineralization
process during OCCM30 cell (mouse cementoblast cells) cultivations [79]. Compared with conventional
process during OCCM30 cell (mouse cementoblast cells) cultivations [79]. Compared with
tissue culture dishes (TCDs), the OCCM30 cells in the fibrin hydrogels had high expression
conventional tissue culture dishes (TCDs), the OCCM30 cells in the fibrin hydrogels had high
levels of biomineralization-associated molecules like bone sialoprotein (BSP), osteocalcin (OCN),
expression levels of biomineralization-associated molecules like bone sialoprotein (BSP), osteocalcin
and Runx-related gene 2 (Runx2) with statistical significances (* p < 0.05; Figure 8). Based on the results,
(OCN), and Runx-related gene 2 (Runx2) with statistical significances (* p < 0.05; Figure 8). Based on
fibrin hydrogel matrices could critically enhance cementogenic or osteogenic differentiation, but the
the results, fibrin hydrogel matrices could critically enhance cementogenic or osteogenic
higher expression level of the plasminogen activator by cementoblasts than by the fibrin matrices
differentiation, but the higher expression level of the plasminogen activator by cementoblasts than
simultaneously led to fibrinolysis (fibrin degradation), loss of cementoblast-fibrin matrix interactions,
by the fibrin matrices simultaneously led to fibrinolysis (fibrin degradation), loss of cementoblast-
and subsequent cell apoptosis in Figure 8 [12,82].
fibrin matrix interactions, and subsequent cell apoptosis in Figure 8 [12,82].

Figure 8. Cementoblast cell activations for biomineralization and fibrinolysis. The fibrin hydrogel
Figure
matrices8. for
Cementoblast cell activations
the cementoblast for could
cell culture biomineralization and fibrinolysis.
activate the canonical The fibrin
Wnt signaling hydrogel
pathway to
matrices for the cementoblast cell culture could activate the canonical Wnt signaling
promote the higher expression of biomineralization-associated molecules like bone sialoprotein (BSP), pathway to
promote the(OCN),
osteocalcin higherand
expression of biomineralization-associated
Runx-related gene 2 (Runx2) (upper panel).molecules like bone sialoprotein
Simultaneously, cementoblast(BSP),
cells
osteocalcin (OCN), and Runx-related gene 2 (Runx2) (upper panel). Simultaneously, cementoblast
could have a significantly high expression level of plasminogen on fibrin hydrogel matrices rather than
cells
tissuecould
culturehave
disha (TCD)
significantly high expression
environments. Moreover,level of plasminogen
expressions on fibrin
of fibrinolytic hydrogel
elements matrices
like matrix
rather than tissue culture
metalloproteinases (MMPs)dish (TCD)during
increased environments. Moreover,
cementoblast expressions
cell cultivation of fibrinolytic
on fibrin hydrogelelements
matrices
like matrix
(lower panel).metalloproteinases
* p < 0.05. Adapted (MMPs) increasedfrom
with permission during cementoblast
the reference [82]. cell cultivation on fibrin
hydrogel matrices (lower panel). * p < 0.05. Adapted with permission from the reference [82].
Moreover, when the fibrin hydrogel was used as the substrate to culture cementoblasts and
Moreover,
osteoblasts when
in vitro, thethe fibrin of
apoptosis hydrogel was used
cementoblasts wasas the substrate
quantitatively to qualitatively
and culture cementoblasts anda
assessed with
osteoblasts
statistically in vitro, thedifference
significant apoptosisfrom
of cementoblasts wasThe
osteoblasts [12]. quantitatively and qualitatively
higher expression level of theassessed with
plasminogen
aactivator
statistically significant difference
by cementoblasts from
rather than osteoblasts
osteoblasts [12].
to the Thematrices
fibrin higher led
expression level of
to fibrinolysis the
(fibrin
degradation), loss of cementoblast-fibrin interactions, and subsequent cell apoptosis [12,82]. Therefore,
 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 17

Int. J. Mol. Sci. 2019, 20, 4364 11 of 17

plasminogen activator by cementoblasts rather than osteoblasts to the fibrin matrices led to
fibrinolysis (fibrin degradation), loss of cementoblast-fibrin interactions, and subsequent cell
apoptosis [12,82].
ε-aminocaproic Therefore,
acid (ACA), ε-aminocaproic acid (ACA),
which is an inhibitor of thewhich is an inhibitor
plasminogen of thewas
activator, plasminogen
utilized to
activator, was utilized to inhibit fibrinolysis and maintain fibrin fibril structures for cementoblast
inhibit fibrinolysis and maintain fibrin fibril structures for cementoblast differentiation, mineralization,
differentiation,
and mineralization,
tissue maturation and tissue
for periodontal tissue regeneration
maturation for
and periodontal
integrations tissue regeneration
[12]. Based on the inand
vitro
integrations [12]. Based on the in vitro findings that modified fibrin hydrogel
findings that modified fibrin hydrogel with ACA molecules, facilitated cementoblast cell with ACA molecules,
viability,
facilitated cementoblast cell viability, and promoted differentiation for mineralization, the in vivo
and promoted differentiation for mineralization, the in vivo periodontal regeneration study was
periodontal regeneration study was designed with fibrin, modified fibrin, and enamel matrix
designed with fibrin, modified fibrin, and enamel matrix derivative (EMD). After the creation of the
derivative (EMD). After the creation of the Class II furcation defect model in beagle dogs, three
Class II furcation defect model in beagle dogs, three different groups were placed to promote cementum,
different groups were placed to promote cementum, PDL, and alveolar bone formations with
PDL, and alveolar bone formations with quantitative and qualitative analyses [12]. Interestingly,
quantitative and qualitative analyses [12]. Interestingly, the modified fibrin matrices could induce
the modified fibrin matrices could induce bone regeneration and mineral deposition on the tooth-root
bone regeneration and mineral deposition on the tooth-root surface, even though only limited PDLs
surface, even though only limited PDLs and orientations of fibrous connective tissues were observed
and orientations of fibrous connective tissues were observed between cementum and bone surfaces
between cementum and bone surfaces (Figure 9) [12].
(Figure 9) [12].

Figure 9. Histological and immunohistological analyses of cementum formation and Sharpey’s fiber
Figure 9. Histological and immunohistological analyses of cementum formation and Sharpey’s fiber
insertions to bone and newly formed cementum tissues. Compared with fibrin-only (unmodified fibrin
insertions to bone and newly formed cementum tissues. Compared with fibrin-only (unmodified
hydrogel) and enamel matrix derivative (EMD) groups, the fibrin-ACP (modified fibrin hydrogel)
fibrin hydrogel) and enamel matrix derivative (EMD) groups, the fibrin-ACP (modified fibrin
facilitated the regeneration of periodontal tissues such as cementum on tooth-root surfaces, PDL, and the
hydrogel) facilitated the regeneration of periodontal tissues such as cementum on tooth-root surfaces,
alveolar bone. More critically, the fibrin-ACP promoted Sharpey’s fiber formations and insertions into
PDL, and the alveolar bone. More critically, the fibrin-ACP promoted Sharpey’s fiber formations and
the cementum layers and alveolar bone surfaces, indicated by white arrows. (d: tooth-root dentin; b:
insertions into the cementum layers and alveolar bone surfaces, indicated by white arrows. (d: tooth-
bone). Adapted with permission from the reference [12].
root dentin; b: bone). Adapted with permission from the reference [12].
Interfaces of cementum-PDL and bone-PDL in the modified fibrin hydrogels showed Sharpey’s
Interfaces of cementum-PDL and bone-PDL in the modified fibrin hydrogels showed Sharpey’s
fiber insertions, which could have fiber anchorages between different mineralized tissue surfaces and
fiber insertions, which could have fiber anchorages between different mineralized tissue surfaces and
re-functionalizing potential as tooth-supportive complexes [12]. It is still hard to demonstrate that the
re-functionalizing potential as tooth-supportive complexes [12]. It is still hard to demonstrate that the
modified fibrin hydrogel could allow the functional restorations of periodontal tissues because the
modified fibrin hydrogel could allow the functional restorations of periodontal tissues because the
structural hierarchies have strongly required mineralized tissue formations with specific orientations of
structural hierarchies have strongly required mineralized tissue formations with specific orientations
PDLs. However, the fibrin hydrogel modification and fabrication could be investigated for regenerative
of PDLs. However, the fibrin hydrogel modification and fabrication could be investigated for
effects for compartmentalized tissue formations with the induction of Sharpey’s fiber formations
regenerative effects for compartmentalized tissue formations with the induction of Sharpey’s fiber
and insertions.
formations and insertions.
Although various scaffolding approaches have been investigated to regulate cementogenesis and
mineralized layer formation with the integration of fibrous connective tissues, the spatial provision for
cementum formation within micron-scaled compartmentalized interfaces is still challenging. Lee et al.
 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 17

Although various scaffolding approaches have been investigated to regulate cementogenesis

Int. J. Mol. Sci. 2019, 20, 4364 12 of 17
and mineralized layer formation with the integration of fibrous connective tissues, the spatial
provision for cementum formation within micron-scaled compartmentalized interfaces is still
challenging.
used Lee et system
the 3D printing al. usedtothe 3D printing
manufacture system to manufacture
multi-layered structures for multi-layered
the periodontalstructures
complex forasthe
well
asperiodontal complex
specific internal poreas well as specific
architectures internal pore
with different architectures
dimensions with different
for individual tissues dimensions for
[59]. In addition,
individual
three tissues
different [59]. In addition,
recombinant three different
human growth recombinantfor
factors (amelogenin human growth factors
cementogenesis, (amelogenin
connective tissue
for cementogenesis,
growth factor (CTGF) for connective tissue growth
PDL regeneration, and bonefactor (CTGF) forprotein-2
morphogenetic PDL regeneration, and bone
(BMP2) for osteogenesis)
morphogenetic
were encapsulated protein-2
using PLGA(BMP2) for osteogenesis)
microspheres spatiallywere encapsulated
tethered using(Figure
to each region PLGA 10) microspheres
[59]. In this
spatially tethered to each region (Figure 10) [59]. In this study, different pore-microstructures
study, different pore-microstructures and types of bioactive molecules in a single tissue engineering and
types of bioactive molecules in a single tissue engineering scaffold strongly correlated
scaffold strongly correlated with multiple tissue regenerations [59]. More interestingly, aligned fibrous with multiple
tissuestructures
tissue regenerations [59]. More
limitedly showedinterestingly,
the fibrousaligned fibrous
insertion and tissue structures
anchorage limitedly showed
to mineralized layers inthethe
fibrous insertion and anchorage to mineralized layers in the PDL region of the scaffold,
PDL region of the scaffold, so the internal pore-structures could be characterized for periodontal so the internal
tissue
pore-structures
integrations could be characterized
and compartmentalized for periodontal
architectures tissue integrations
with bioactive molecules couldand compartmentalized
lead to the formation
architectures with bioactive molecules could
of fibrous-mineralized tissue constructs (Figure 10) [59].lead to the formation of fibrous-mineralized tissue
constructs (Figure 10) [59].

Figure
Figure10. 10. Optical
Optical andand scanning electron microscopic
scanning electron microscopic (SEM)
(SEM)images
imagesofof3D 3Dprinted
printedscaffold
scaffoldandand
histological analyses. Specific pore dimensions with the spatial compartmentalization
histological analyses. Specific pore dimensions with the spatial compartmentalization were designed were designed
for
formultiple
multipletissue
tissueformation
formationand and infiltration,
infiltration, such
such asas cementum,
cementum,periodontal
periodontalligament
ligament(PDL),
(PDL),andandthethe
alveolar
alveolarbone.
bone. In In particular, poly(lactic-glycolic) acid
particular, poly(lactic-glycolic) acid (PLGA)
(PLGA)microspheres
microsphereswere weretethered
tetheredwith
with
different
differentbioactive
bioactivemolecules
moleculesto to promote
promote individual tissue growth
individual tissue growthlike
likethe
theamelogenin
amelogeninfor forcementum,
cementum,
the
theconnective
connectivetissue
tissuegrowth
growthfactor
factor(CTGF)
(CTGF)for forPDL,
PDL,and bone
and bonemorphogenetic
morphogenetic protein-2
protein-2(BMP2)
(BMP2) forfor
the
alveolar bone. The histology analyses with hematoxylin and eosin (H&E) staining
the alveolar bone. The histology analyses with hematoxylin and eosin (H&E) staining and Alizarin and Alizarin red
staining showed
red staining showedthe mineralized
the mineralized tissue formation
tissue formation and
andfibrous
fibroustissue
tissue insertion intothe
insertion into themineralized
mineralized
tissues
tissuesininvivo,
vivo,which
whichwas wasperformed
performed with with the
the subcutaneous immunodeficientmouse
subcutaneous immunodeficient mousemodel
model[59].
[59].

4.4.Future
FutureProspects
Prospectsfor
forBiomaterial-Based Periodontal Tissue
Biomaterial-Based Periodontal Tissue Engineering
Engineering
3D3Dprinting techniques
printing techniqueshavehave
beenbeen
employed to develop
employed various various
to develop scaffolding designs for
scaffolding periodontal
designs for
tissue regeneration for pre-clinical and clinical scenarios to spatiotemporally compartmentalize
periodontal tissue regeneration for pre-clinical and clinical scenarios to spatiotemporally
individual tissues such
compartmentalize as cementum,
individual tissues PDL,
suchand
as the alveolar PDL,
cementum, bone [30–32].
and the However, the technology
alveolar bone [30–32].
is still challenged to create micron-scaled tissue compartments for the individual periodontium
(especially, cementum and PDLs) and to control spatiotemporal tissue formations with their unique
individual characteristics [5,37]. This review highlights the recently developed 3D platforms to guide
Int. J. Mol. Sci. 2019, 20, 4364 13 of 17

orientations of engineered PDLs and promote cementogenesis as the interfacial tissue layer to induce
tissue integrities by biomaterial modifications for the revitalization of periodontal complexes in vitro
and in vivo. Although these novel techniques need to integrate existing techniques like 3D printing,
cell sheet engineering, or cell spheroid approaches, they will be the principal and predominant
strategies for the new paradigm of periodontal regenerative medicine with greater predictability and
high controllability.

Author Contributions: C.H.P. outlined and wrote the manuscript as well as designed figures.
Funding: This work was supported by Osteology Foundation (#16-173) and National Research Foundation of
Korea (NRF-2016R1D1A1B03935686).
Conflicts of Interest: The author declares no conflict of interest.

Abbreviations
PDL Periodontal ligament
CEJ Cementoenamel junction
GTR Guided tissue regeneration
3D 3-dimensional
Micro-CT Micro-computed tomography
SEM Scanning electron microscopy
PCL Poly-ε-caprolactone
H&E Hematoxylin and eosin
PCE Poly-ε-caprolactone and polyethylene glycol
CVD Chemical vapor deposition
TCD Tissue culture dish
BSP Bone sialoprotein
OCN Osteocalcin
Runx2 Runx-related gene 2
MMPs Matrix metalloproteinases
EMD Enamel matrix derivative
ACA ε-aminocaproic acid
ACP ε-aminocaproic acid incorporated with chitosan nanoparticle
CaP Calcium phosphate
CAP Cementum attachment protein
CEMP Cementum protein
PLGA Poly(lactic-co-glycolic acid)
CTGF Connective tissue growth factor
BMP-2 Bone morphogenetic protein-2
PDGF-BB Platelet-derived growth factor-BB

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