Tooth Repair and Regeneration

Abstract

Purpose of Review
Current dental treatments are based on conservative approaches, using inorganic materials and appliances.
This report explores and discusses the newest achievements in the field of “regenerative dentistry,” based on the concept of biological repair as an alternative to the current conservative approach.

Recent Findings
The review covers and critically analyzes three main approaches of tooth repair: the re-mineralization of the enamel, the biological repair of dentin, and whole tooth engineering.

Summary
The development of a concept of biological repair based on the role of the Wnt signaling pathway in reparative dentin formation offers a new translational approach into development of future clinical dental treatments.
In the field of bio-tooth engineering, the current focus of the researchers remains the establishment of odontogenic cell-sources that would be viable and easily accessible for future bio-tooth engineering.

Full text

DENTAL STEM CELLS IN TISSUE REGENERATION (F SETZER, SECTION EDITOR)
Tooth Repair and Regeneration
Ana Angelova Volponi
1
&Lucia K. Zaugg
1,2
&Vitor Neves
1
&Yang Liu
1
&Paul T. Sharpe
1
Published online: 25 October 2018
#The Author(s) 2018
Abstract
Purpose of Review Current dental treatments are based on conservative approaches, using inorganic materials and appliances.
This report explores and discusses the newest achievements in the field of “regenerative dentistry,”based on the concept of
biological repair as an alternative to the current conservative approach.
Recent Findings The review covers and critically analyzes three main approaches of tooth repair: the re-mineralization of the
enamel, the biological repair of dentin, and whole tooth engineering.
Summary The development of a concept of biological repair based on the role of the Wnt signaling pathway in reparative dentin
formation offers a new translational approach into development of future clinical dental treatments.
In the field of bio-tooth engineering, the current focus of the researchers remains the establishment of odontogenic cell-sources
that would be viable and easily accessible for future bio-tooth engineering.
Keywords Regenerative dentistry .Dentinogenesis .Bio-tooth .Biological repair .Reparative dentin
Introduction
Tooth loss is a global health problem representing a burden to
society and the economy [1]. It affects an individual’s capacity
for biting, chewing, smiling, speaking, and psychosocial
wellbeing. Complete loss of natural teeth is widespread, partic-
ularly affecting older people [2,3]. Dental caries, periodontal
disease, and genetic disorders are major causes of tooth loss.
Caries is reported as the most common disease worldwide
[4,5]. Current dental treatments used to replace missing tooth
structure or missing teeth are based on conservative therapies
such as fillings, made of inert dental materials, fixed dental
bridges, or removable dentures and dental implants.
Osseointegrated dental implants revolutionized dentistry as
they provide restoration of lost function without affecting
healthy teeth [6]. Although current dental implants mark
notable advantages in osseointegration and soft tissue adapta-
tions, the concept of the treatment is based on the usage of
inert materials in direct contact with bone tissue and absence
of periodontal ligament (PDL) tissue. The PDL physiological-
ly provides a buffered distribution of mastication forces and
when absent can often lead to jaw bone resorption [7,8].
Regenerative dentistry is an emerging concept that challenges
the modern dentistry to step up the dental research and translate
the scientific knowledge into new future clinical treatments.
The approach is based on understanding the underlying
mechanisms of tooth development and the biological process-
es of healing and repair, creating a solid knowledge of prin-
ciples that could be applied in harnessing the natural healing
potential of the dental tissues, or regenerating (engineering)
the damaged tissue or organ.
Healing and Engineering Different Dental
Tissues
Biomimetic Approach in Repairing the Damaged
Enamel
Repairing a Cell-Free Tissue
A tooth is a complex organ consisting of a soft connective tissue
(dental pulp) encased in a chamber of differently mineralized
This article is part of the Topical Collection on Dental Stem Cells in
Tissue Regeneration
*Ana Angelova Volponi
ana.angelova@kcl.ac.uk
1
Centre for Craniofacial and Regenerative Biology, Dental Institute,
King’s College London, London, UK
2
Department of Periodontology, Endodontology and Cariology,
University Center for Dental Medicine Basel, University of Basel,
Basel, Switzerland
Current Oral Health Reports (2018) 5:295–303
https://doi.org/10.1007/s40496-018-0196-9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

hard tissues (enamel, cementum, and dentin). The outer miner-
alized tissue in the crown region, the enamel, is the highest
mineralized tissue of the human body, characterized by an ab-
sence of cells. It provides the first hard barrier towards the outer
environment, protecting the tooth from damage.
During tooth development, the ameloblasts, which are re-
sponsible for the formation of enamel, undergo programmed
cell death at the maturation stage and no longer exist in the
mature enamel [9].
Therefore, once damaged, enamel cannot be biologically
regenerated/repaired. Hence, the concept of “healing”of the
damaged enamel consists in repairing by acellular re-minerali-
zation. Traditionally, fluoride (F) and calcium phosphate
nanocrystals are applied to re-mineralize the eroded enamel
matrix and act by inhibiting demineralization by fluoride incor-
poration in the crystal lattice, resulting in lower solubility of
enamel [10], and having a potential to protect the outer ~
30 μmofthetooth[11]. The newly formed hydroxyapatite
usually lacks the structure and mechanical properties of the
natural enamel [12,13]. Therefore the biggest challenge lies
in recreating the hierarchical structure on the surface of the
damaged enamel. The unique cross-arranged structure of enam-
el exhibits two important components: prismatic and
interprismatic areas, which have different stabilities to resist
acid erosion [14]. So, the challenge remains to synthesize apa-
tite nanocrystals with a proper oriented structure, similar to the
natural enamel, directly on the surface of the damaged enamel
andinanoralenvironment[15,16].
Recently, different biomimetic approaches have been de-
veloped to synthesize artificial dental enamel. In a study by
Kind L. et al. [17], self assembling peptides were used to
facilitate the subsurface mineralization of the enamel in cari-
ous lesion, while other groups used elastin-like polypeptide-
assisted biomimetic approach to synthesize artificial dental
enamel (Fig. 1a) [16]. Although these materials act only on
the surface of enamel demineralization at present, enamel-
oriented growth sheds a light on the potential of structural
and mechanical regeneration of enamel cavitation.
Still, a major goal in the fast-developing material science
remains creation, design, and development of bioinspired
functional structures, using synthetic hierarchical materials
with enhanced functionality, like the dental enamel [18•].
Moreover, the open question remaining is the time needed
for enamel remineralization to form a functional enamel tis-
sue. This issue will lastly determine the viability of this ap-
proach for a clinical treatment.
Biological Repair of the Dentine-Pulp Complex
The dental pulp is a soft connective tissue, containing different
cell populations, which is encased in a thick, porous mineral-
ized chamber. As a clinical approach, keeping the vitality of
the dental pulp has always been the goal for a successful long-
term restorative dental treatment. In the adult pulp, cell divi-
sion and the secretory activity of odontoblasts decrease in
comparison to the developing stage; however, these processes
are re-activated following pulp damage.
In shallow enamel and enamel/dentinal damage, odontoblasts
can survive, and its activation supports repair, protecting the
dental pulp via reactionary dentine formation [19,20].
However, in situations of deep cavitation or trauma that involves
dental pulp exposure, odontoblasts may not survive and will
have to be replaced, requiring a cascade of stem cell activation,
proliferation, and differentiation into new odontoblast-like cells
that will culminate into reparative dentine secretion [21].
Because of the capacity of teeth to repair themselves and their
accessibility, Gronthos and collaborators [22] researched and
identified a population of new cells isolated from the dental pulp
of human third molars. They termed these cells dental pulp stem
cells (DPSCs) and showed that these cells produce dentine
in vitro [23]. Although these data show that dental pulp cells
have the capacity to repair damaged dental tissues, when it
comes to in vivo tooth repair, the dental reparative capacity is
limited to the critical size of the damage [24,25]. In large,
exposed pulpal injuries, the pulp is exposed to microorganisms
from the oral cavity, and if the infected dentine is not completely
removed before applying a biocompatible material while direct-
ly capping the exposed pulp, the dental pulp will undergo ne-
crosis. This highlights the clear need to generate a therapy that
stimulates the full biological potential the dental pulp has to
repair injuries comprising dentine and pulp.

Fig. 1 Schematic representation of different approaches for dentine-pulp
complex repair/regeneration (a) and stem-cell-based whole-tooth
bioengineering (b). aPharmacological modulation of Wnt/β-catenin
signaling pathway shows natural dentine apposition in both, deep
cavitation without pulp exposure (reactionary dentine, upper left box)
and cavitation with exposed pulp tissue (reparative dentine, upper right
box), as long as the underlying pulp tissue is vital and harbors resident
odontoblasts and dental pulp stem cells (DPSCs) respectively. The simple
applicability of this technique by using a drug-enriched collagen sponge
makes it ideal for a translational clinical treatment approaches. In case of
pulp infection and necrosis (lower right box), current therapies include
orthograde root canal treatment or, in selected cases with incomplete root
formation, revascularization procedures. Recent cell-based approaches
show that autologous isolated, expanded, and mobilized DPSCs have
the capacity to re-innervate (positive response on pulp testing) a
pulpectomized and disinfected tooth after auto-transplantation; however,
this approach is highly technique sensitive and might remain in facilities
with specialized equipment and laboratories for selected cases only. Non-
cell-based approaches for mimicking lost enamel-structure exists (lower
left box) yet the mineralization potential of self-assembling peptides
needs to be further evolved and clinically tested. bSuitable adult
sources of epithelial and mesenchymal cells are collected from the
patients with missing teeth and expanded in vitro. Either epithelial or
mesenchymal cell populations are induced to be odontogenic (capable
of initiation of de novo odontogenesis) and recombined with the
responsive cell population counterpart. An early-stage tooth
primordium can be generated from the epithelial-mesenchymal-cell
recombination, which can be subsequently either directly transplanted at
the location of the missing tooth or cultured ex vivo to form a whole tooth
to replace the missing tooth
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Reactionary Dentin
During primary and secondary dentin deposition, odontoblasts
secrete growth factors and proteins that are fossilized in the den-
tine matrix after mineral maturation [26]. As decay demineralizes
the dentine, these growth factors and proteins are released to the
dental pulp to activate odontoblasts, immune cells, and organize
the reactionary dentine matrix secretion [27]. Hydraulic silicate
cements, e.g., mineral trioxide aggregate (MTA), Biodentine,
total fill, endosequence, calcium-enriched cement etc., claim to
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be bioactive. As demonstrated by Loison-Robert and colleagues
[28], Biodentine promotes mineralization when in contact with
dental pulp stem cells (DPSCs); however, it does not affect
DPSC proliferation. Interestingly, the only biological factor that
Biodentine has shown to increase is TGF-β1[29]; however,
Neves and Sharpe [30••] showed that TGF-βand BMP are not
pivotal for reactionary dentine formation but for tubular organi-
zation of reactionary dentine secretion. Reactionary dentine se-
cretion is affected, however, by changes in the Wnt pathways.
Wnt activation via GSK-3 inhibitor drugs increased reactionary
dentine secretion, although Wnt inhibition did not impair reac-
tionary dentine secretion (Fig. 1a) [30••].
One clinical specialty that could benefit from the advances of
a reactionary dentine promoting material is prosthetic dentistry.
The creation of a material that cements a crown and stimulates
reactionary dentine formation might change the prosthetic plan-
ning approach, as it increases the rate of pulp vitality post tooth
preparation to receive a crown. Biomaterials with the capacity to
incorporate Wnt activators, such as Bioglass, have been devel-
oped [31], but further testing is still to be done.
Reparative Dentine
When a dental injury reaches the dental pulp, a more complex
clinical and biological approach is taken to preserve pulp vitality
and reconstruct the lost dentine. Studies have revealed that den-
tine matrix derivatives and breakdown products from the dental
pulp influence pulp cell migration. Recruited cells exhibited in-
creased stem cell marker expression indicating that dental ECMs
and their breakdown products selectively attract progenitor cells
that contribute to repair processes [25,32]. These mobilized res-
ident dental pulp mesenchymal stem cells differentiate into new
odontoblast-like cells that secrete a form of tertiary (reparative)
dentine [33•,34•,35–37]. This dentine is laid in a form of a thin
band of dentine (dentine bridge) that walls off the pulp from
bacterial infection. Studies have focused in understanding the
underlying mechanisms of this process of “natural healing,”in
order to learn how to pharmacologically trigger this event for
promoting reparative dentine formation [34•].
A recent preclinical study showed that small molecules deliv-
ered via a biodegradable collagen sponge were able to provide an
effective repair of experimentally induced deep dental lesions by
stimulating Wnt/β-catenin signaling and hence promote repara-
tive dentine formation [38]. This study was based on the natural
underlying mechanisms and pathways that are pivotal for the
healing mechanisms of the dental pulp. The activation of Wnt/
β-catenin signaling is an immediate early response to tissue dam-
age; moreover, Wnt receiving cells become odontoblast-like
cells, proving they are stem cells [34•,38–42]. After confirming
that Wnt/β-catenin is upregulated following tooth damage, the
study showed that an addition of small-molecule Wnt agonists,
tested in human clinical trials for Alzheimer, can stimulate the
dental natural response, triggering reparative dentine formation
and thus restoring the lost dentine structure with naturally gener-
ated new dentine. The striking “simplicity”of this technique
makes it ideal for a translatable clinical technique; also, it helps
opening a new door for more “biomimetic”approaches aiming to
repair the dentine-pulp complex naturally (Fig. 1a). The fact that
these particular small-molecule Wnt agonists are undergoing
clinical trials already contributes further to the potential of trans-
lation into future dental treatments through an adequate incorpo-
ration into a dental material as a carrier.
When microorganisms or their endotoxins reach the dental
pulp and it is diagnosed with irreversible pulpitis, because this
condition cannot be reversed, the common treatment for this
situation has been pulpectomy, regardless of the amount of the
remaining unaffected pulp tissue. With the standard protocol, the
entire pulp has to be removed, followed by root canal treatment,
disinfecting the pulp space and replacing it with inorganic mate-
rials [43]. However, new approaches on de novo regeneration of
the dental pulp have been investigated and will be further
discussed below.
De Novo Regeneration of Dental Pulp
An approach for repairing/regenerating the lost dental pulp tis-
sue, following a root canal treatment is the “de novo”regener-
ation of dental pulp tissue. Currently, clinical procedures are
postulated to successfully revascularize infected teeth with in-
complete root formation to achieve full root length development
and dentine thickness. A variety of promising cases have been
published that indicated pulp revascularization with pulp- and
dentin-like tissue formation and healing of apical inflammation
[44–46]. However, this procedure is limited to infected or non-
infected immature teeth with open apices [47]. To address tissue
regeneration in fully formed teeth, procedures based on cellular
approaches should be further considered and developed. Among
the potential sources of stem cells isolated from teeth (DPSCs,
stem cells from human exfoliated deciduous teeth [SHED] and
stem cells of the apical papilla [SCAP]), Cordeiro and col-
leagues [48] suggested that SHED was the most valuable cell
source for dental pulp tissue engineering due to its capacity to
generate pulp tissue in human tooth slices with similar architec-
ture to those of a physiologic dental pulp.
In 2010, Huang et al. [49] used different dental stem cells
isolated from human dental pulp of permanent teeth (DPSCs)
and stem cells isolated from the apical papilla region of human
third molars (SCAP cells), to demonstrate de novo regeneration
of dental pulp in empty root canal spaces. These cells were seed-
ed onto poly-D,L-lactide/glycolide scaffold and inserted into the
canal space of root fragments followed by subcutaneous trans-
plantation into immunocompromised-SCID mice. After a period
of 3 to 4 months, a histological analysis revealed that the root
canal space was filled with pulp-like tissue with well-established
vascularization. Moreover, a continuous layer of mineralized tis-
sue resembling dentine was deposited on the existing dentinal
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walls of the canal. This dentine-like structure appeared to be
produced by a newly formed layer of odontoblast-like cells [49].
In a more advanced attempt to regenerate whole dental pulp
in a real clinical scenario, a recent clinical pilot study in humans
assessed the therapeutic potential and safety of mobilized dental
pulp stem cells (MDPSCs) to regenerate the dental pulp de
novo in teeth that suffered from irreversible pulpitis without
any periapical lesions [50,51]. DPSCs were isolated from a
small amount of pulp tissue of autologous discarded teeth using
a granulocyte colony stimulating factor (G-CSF)-induced mo-
bilization method. MDPSCs were successfully transplanted in
previously disinfected, empty root canal spaces in vivo.
Magnetic resonance imaging (MRI) and cone beam computed
tomography (CBCT) revealed changes in the dental pulp indi-
cating a pulp-like regenerated tissue and new dentine apposition
after 24 weeks in most cases with positive response to electrical
pulp testing (Fig. 1a) [50,51].
While these studies focused on the usage of different dental
stem cells in dental pulp tissue engineering, other studies focused
on exploring new mechanical systems as options for scaffolds in
pulp regeneration [52]. New injectable microsphere systems,
where vascular endothelial growth factor (VEGF) binds with
heparin and is encapsulated in heparin-conjugated gelatine nano-
spheres of a biodegradable poly L-lactic acid (PLLA) micro-
sphere, mimic natural ECM-like collagen structures and hence
act as carrier for DPSC leading to pulp tissue formation by pro-
moting their proliferation/differentiation. Additionally, these sys-
tems provide a controlled release of the VEGF resulting in newly
formed blood vessels within the regenerated tissue [52,53].
Over the last decades, conservative endodontic procedures
focused on techniques to enhance root canal disinfection and
irrigation in order to address infection control of complex ana-
tomical structures like isthmuses, and lateral canals or the apical
delta. Since the presence of microbesinsuchareascancontrib-
ute to persistent inflammation, all procedures of de novo regen-
eration need to follow a procedure of complete root canal dis-
infection. From a clinical perspective, all the above-mentioned
cellular-based cases of de novo pulp regeneration have been
performed in teeth with relatively simple root canal anatomy
and rather large canals. Hence, clinical applicability of teeth
with more complex root canal anatomy may be questioned.
The idea of supporting the seeded dental stem cells, in this
case dental pulp stem cells from adult teeth (DPSCs), provid-
ing a three-dimensional, controlled environment with added
growth factors, emphasizes once more the underlying concept
of the “regenerative”approach, where the biological system is
mimicked and recreated in its complexity.
Whole-Tooth Bioengineering
Whole-tooth bioengineering has always been the ultimate goal of
regenerative dentistry. (Fig. 1b) Despite recent progress in this
field [54–56,57••,58], we are still facing a number of difficult
challenges to overcome. The basic principle of this “organ engi-
neering”approach is understanding the mechanisms that regulate
the embryonic tooth development and recreating these events
in vitro, mimicking the natural cascade of signaling that occurs
during organ formation [57••].
Due to its non-essential function and accessibility, it
represents an important model to study organogenesis. In
common with other ectodermal appendages, like hair fol-
licles and exocrine glands (mammary, sweat, and salivary),
tooth morphogenesis is guided by reciprocal interactions
between epithelial and mesenchymal tissues and pro-
gresses through distinct stages [59,60]. The knowledge
gained in bio-tooth engineering potentially could have a
broader impact in the field of regenerative medicine and
the repair of different organs.
Mimicking the Natural Events of Tooth Development
Several decades ago, classical tissue recombination exper-
iments demonstrated sequential signaling between the den-
tal epithelium and the mesenchyme of different origins and
stages, where the epithelium acted as an inductive tissue
[61,62]. This odontogenic induction in the epithelium is
lost early, as it naturally happens during the embryonic
tooth development and then switches to mesenchyme.
Thus, the mesenchyme becomes the odontogenic inductive
source.
In 2003, Yamamoto and collaborators [63]showedthe
ability of embryonic tooth germ cells to re-aggregate fol-
lowing dissociation and form teeth. Other studies followed
where epithelium and mesenchyme tissues from E14.5- to
E12.5-stage mouse tooth germs were separated and the
cells dissociated and recombined to form normal teeth
[64–67].
This reciprocal tissue induction that takes place during
the early stages of tooth development, whereby the epi-
thelium first induces tooth formation in the mesenchyme
followed by a reciprocal induction from mesenchyme to
epithelium, has been utilized to suggest a basis for
whole-tooth bioengineering that could employ adult cells
[59,68,69].
In 2004, a study by Ohazama et al. [69] showed that when
mesenchyme cells derived from adult bone marrow are com-
bined with inductive-stage embryonic dental epithelium, tooth
formation is induced, and the adult mesenchymal cells respond
and fully contribute to tooth development [69]. Another study in
2013 [70] showed that human gingival epithelial cells were able
to respond to the inductive signal of mouse tooth embryonic
mesenchyme, resulting in formation of fully formed teeth.
However, in spite of the fact that adult stem cells can re-
spond to an inductive odontogenic signal and participate in
tooth formation, the only cells that have been shown to be
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capable of tooth-inductive capacity are odontogenic embryon-
ic cells, derived from inductive embryonic tooth-germ tissue
(epithelium or mesenchyme) [54–56,57••,58]. Furthermore,
in all experiments reported to date, the inductive cells, whether
epithelial or mesenchymal, do not retain their inductive capac-
ity following in vitro expansion [71]. This defines one of the
biggest challenges in the field of biotooth engineering, which
is to identify adult cell populations that retain their
odontogenic potential and can be expanded in large numbers.
Moreover, these cells should ideally be allogeneic, where one
population, either mesenchyme or epithelium, has tooth-
inducing capacity, avoiding any issues that use of non-
allogeneic cells may have for generation of nonessential or-
gans such as teeth, in a clinical treatment.
Maintaining the Odontogenic Potential
In Vitro
Three-Dimensional Microenvironmental
Reprogramming
The arrangement of cells within a tissue plays an essential role
in organogenesis, including tooth development [72]. In the
condensed mesenchyme, during the tooth development, cells
change their shape and size dynamically.
The size and shape of the condensed cell mass also dictate
the final three-dimensional form of the organ, and abnormal
condensation can result in developmental defects [73].
Mechanical stimuli can modulate cell lineage commitment
and control development of various tissues during embryo-
genesis, and studies with cultured cells suggest that cell fate
can be controlled mechanically by altering cell shape [74,75].
These observations raise the possibility that physical alter-
ations in cells that result from cell compaction in the con-
densed mesenchyme also could play an active role in the dif-
ferentiation process [73].
In order to preserve the odontogenic signal in cells that
have been expanded in vitro,Kuchler-Bopp et al. [72], have
proposed a three-dimensional micro-culture system, where
they tested the hanging drop method to study mixed
epithelial-mesenchymal cell reorganization in a liquid medi-
um, showing that the system offers the microenvironmental
conditions for tooth histogenesis and organogenesis. It was
shown that this method can provide control of the proportion
and number of cells to be used, and the forming micro tissues
showed homogeneous size.
Cell Community Effect in Preserving the Odontogenic
Potential
In 2016, Yang and colleagues [57••] proposed another ap-
proach to preserve the odontogenic signals in embryonic tooth
germ cells that have rapidly lost their tooth-inducing capacity,
once expanded in vitro. The study suggested that uncultured
embryonic tooth germ mesenchymal cells were able to rescue
cultured cells and enable them to fully participate into
bioengineered tooth development, giving rise to dental pulp
cells and odontoblasts. Although this rescue effect was not
observed with postnatal dental pulp mesenchyme cells, this
finding indicates that the presence of fresh (non-cultured) em-
bryonic tooth germ cells can have a “community effect,”iden-
tified during embryonic development as a process that enables
mixtures of different cells to differentiate along the same
pathway.
Conclusion
The newest trends in the dental research field propose a con-
cept of regenerative dentistry, based on harnessing the natural
healing abilities of the dental tissues through biological repair.
Although major steps are achieved in creating synthetic hier-
archical materials that could be used in re-mineralization of the
enamel, the enhanced functionality and the time for the re-
mineralization of a tissue as the enamel remains an obstacle.
New data suggest that by mobilizing the stem cells through
the Wnt signaling pathway—a particular cascade of molecules
involved in cell-to-cell communication (essential for tissue
repair and stem cell development), formation of reparative
dentin can be achieved.
Moreover, usage of an already clinically tested drug known
to stimulate Wnt signaling investigated in clinical trials for its
potential to treat Alzheimer’s and other neurological disorders
opens the possibility of a fast clinical translation for future
biological dental treatments of dental cavities.
New research work contributed to the understanding of the
underlying mechanisms of tooth development, mimicking the
events in vitro,as an approach of bio-tooth engineering. The
current focus of the researchers remains establishment of easily
accessible cell sources that would maintain the odontogenic sig-
nal after in vitro expansion for future bioengineering.
Compliance with Ethical Standards
Conflict of Interest Dr. Sharpe reports a patent pending. All other au-
thors declare no conflicts of interest.
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
the authors.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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