biomimetics

Review
Biomimetic Aspects of Oral and
Dentofacial Regeneration
Akshaya Upadhyay † , Sangeeth Pillai † , Parisa Khayambashi, Hisham Sabri , Kyungjun T. Lee,
Maryam Tarar, Stephanie Zhou, Ingrid Harb and Simon D. Tran *
McGill Craniofacial Tissue Engineering and Stem Cells Laboratory, Faculty of Dentistry, McGill University,
3640 University Street, Montreal, QC H3A 0C7, Canada; akshaya.upadhyay@mail.mcgill.ca (A.U.);
sangeeth.pillai@mail.mcgill.ca (S.P.); parisa.khayambashi@mail.mcgill.ca (P.K.);
hisham.sabri@mail.mcgill.ca (H.S.); kungjun.lee@mail.mcgill.ca (K.T.L.); maryam.tarar@mail.mcgill.ca (M.T.);
stephanie.zhou@mail.mcgill.ca (S.Z.); ingrid.harb@mail.mcgill.ca (I.H.)
* Correspondence: simon.tran@mcgill.ca
† These authors contributed equally to this work.




Received: 15 September 2020; Accepted: 10 October 2020; Published: 12 October 2020


Abstract: Biomimetic materials for hard and soft tissues have advanced in the fields of tissue
engineering and regenerative medicine in dentistry. To examine these recent advances, we searched
Medline (OVID) with the key terms “biomimetics”, “biomaterials”, and “biomimicry” combined with
MeSH terms for “dentistry” and limited the date of publication between 2010–2020. Over 500 articles
were obtained under clinical trials, randomized clinical trials, metanalysis, and systematic reviews
developed in the past 10 years in three major areas of dentistry: restorative, orofacial surgery,
and periodontics. Clinical studies and systematic reviews along with hand-searched preclinical studies
as potential therapies have been included. They support the proof-of-concept that novel treatments
are in the pipeline towards ground-breaking clinical therapies for orofacial bone regeneration,
tooth regeneration, repair of the oral mucosa, periodontal tissue engineering, and dental implants.
Biomimicry enhances the clinical outcomes and calls for an interdisciplinary approach integrating
medicine, bioengineering, biotechnology, and computational sciences to advance the current research
to clinics. We conclude that dentistry has come a long way apropos of regenerative medicine; still,
there are vast avenues to endeavour, seeking inspiration from other facets in biomedical research.

Keywords: biomimetics; dentistry; dentofacial; regeneration

1. Introduction
The term “biomimetics” was derived from the Greek words “bios” (meaning life) and “mimesis”
(meaning to imitate). It originally meant developing any new material or technology that mimics
nature or is obtained from nature. In biology, biomimetics relates to harnessing bioinspired materials or
molecules, either synthetic replacements of natural structures or derivations from living organisms that
simulate biological mechanisms. The field of tissue engineering and regenerative medicine (TERM)
has developed significantly over the past decade with the main focus on the synthesis of novel, highly
intricate biomaterials and techniques to regenerate and replace lost structures. However, in light of
the human body’s complex anatomy and functions, it has always remained a challenge to develop
state-of-the-art, accurate, bio-replacements for different tissues and organs. The craniofacial region is
residence to living paradoxes, with hardest to the softest tissues, and involves a macromolecular to
nanomolecular range of therapies. Each tissue and organ has their own peculiarities that have to be dealt
with to achieve the utmost biological resemblance to natural tissues. Biomimetic dentistry has come a
long way in engineering and regenerating dental hard and soft tissues unprecedentedly. We hypothesize

Biomimetics 2020, 5, 51; doi:10.3390/biomimetics5040051 www.mdpi.com/journal/biomimetics

Biomimetics 2020, 5, 51 2 of 45

that biomimetic improvements are highly essential in successfully engineering dentofacial structures.
This current review provides a glimpse of the volumes of work that has been done over the past
decade to improve these aspects. The purpose of this study is to establish a guide to new researchers,
clinicians, and dentists at all stages of research to help them develop a perspective of biomimetics and
its importance in clinical therapies, specifically in restorative dentistry, oral and maxillofacial surgery,
and periodontology.
The literature was searched on Medline (OVID) with key terms like “biomimetics”, “biomaterials”,
and “biomimicry” combined with MeSH terms for “dentistry”, and a limit was set to 2010–current,
where over 500 articles were obtained under clinical trials, randomized clinical trials, metanalysis,
and systematic reviews together. Out of the obtained search, relevant clinical studies have been
included with systematic reviews, along with hand-searched preclinical studies as potential therapies
wherever deemed necessary. A brief discussion on enamel regeneration and recent outlooks in pulp
regeneration has been covered. Pulp tissue is a complex connective tissue with multiple functions
including protective, nutritive, and reparative activities. Infection to pulp due to caries or trauma
usually results in complete pulp removal. This makes the tooth structure more fragile and prone to
fracture. Therefore, it is important to use the most biomimetically advanced material in these situations
to preserve or replace the pulp tissue to save the tooth structure. We have discussed the most significant
biomimetic analogues for tooth structure and different scaffolds for dentin pulp complex regeneration.
Further, we have focused on the current preclinical and clinical studies in orofacial bone regeneration
and a brief overview of strategies for repair of the oral mucosa under oral and maxillofacial dentistry.
As important as the tooth structure, it is also essential to understand the supporting periodontal
structure, which are the hard and soft tissue that surround the tooth that undergoes competitive
regeneration processes. An array of different tissue engineering strategies and surgical methods are
used today to induce regeneration of the complex highly cellular periodontium, and thus, in the last
section of this review, we cover the current facets in periodontal tissue engineering, including cell and
cell-free approaches and guided tissue regeneration, with a brief description of implant biomimetics.

2. Biomimetics in Restorative Dentistry

2.1. Enamel Biomimetics

Enamel is the outermost hard tissue covering the crown of the tooth structure. It is considered the
hardest substance in the body due to its high inorganic content (96%), mostly comprised of interwoven
hydroxyapatite crystals arranged in a three-dimensional pattern, giving it superior aesthetic and
structural properties [1]. However, continuous and complex changes occurring within the oral
microenvironment sometimes lead to enamel demineralisation, thereby initiating caries formation.
Dental caries affects more than two-thirds of the world’s population and is highly prevalent among
people of all ages. The origination of caries is contributed by a multitude of factors including the
presence of cariogenic bacteria, dietary carbohydrates, decreased salivary flow, or xerostomia. Usually,
there is a balance between the demineralisation and remineralisation processes in the oral cavity,
but this equilibrium is lost due to factors consistently favouring tooth demineralisation, leading to
primary white spot lesions, caries progression, and eventually cavitation [2]. Proper teeth cleaning
to get rid of cariogenic bacteria, adequate salivary flow, and the presence of sufficient amounts of
calcium and phosphate ions in saliva can help control the limit of tooth demineralisation to a certain
extent. However, since the body’s natural defence might not be enough to resist caries, in many cases,
minimally invasive dentistry approaches are used in a desperate attempt to remove initial caries and
to preserve as much of the natural tooth structure to maintain the functional integrity and aesthetics
of the tooth. Nevertheless, enamel regeneration still remains a challenging task, and it becomes
even more complex on clinical implementation. Therefore, it is essential to look at alternate methods
for enamel repair and engineer biomaterials that mimics the natural enamel both biologically and
structurally. Pandya et al. (2019) described four different pathways for enamel tissue engineering
Biomimetics 2020, 5, 51 3 of 45
Biomimetics 2020, 5, 51 3 of 46

pathways for enamel tissue engineering and regeneration (Figure 1) by (a) physiochemical synthesis,
and regeneration (Figure 1) by (a) physiochemical synthesis, (b) protein-matrix-guided enamel crystal
(b) protein-matrix-guided enamel crystal development, (c) enamel surface remineralisation, and (d)
development, (c) enamel surface remineralisation, and (d) cell-based regeneration [3]. We will discuss
cell-based regeneration [3]. We will discuss these approaches with their most recent advances in
theseenamel
approaches with their most recent advances in enamel mimetics.
mimetics.

Figure
Figure 1. Mechanisms
1. Mechanisms of of enameltissue
enamel tissue engineering
engineering andand regeneration: (a) physiochemical
regeneration: synthesis
(a) physiochemical of
synthesis
apatite crystals, (b) protein-matrix-guided enamel crystal development, (c)
of apatite crystals, (b) protein-matrix-guided enamel crystal development, (c) enamel surface enamel surface
mineralisation using fluoride toothpastes, and (d) ameloblast (cell-based) tissue engineering of
mineralisation using fluoride toothpastes, and (d) ameloblast (cell-based) tissue engineering of synthetic
synthetic enamel apatite. Image adapted from [3] Pandya, M.; Diekwisch, T.G.H. Enamel
enamel apatite. Image adapted from [3] Pandya, M.; Diekwisch, T.G.H. Enamel biomimetics-fiction or
biomimetics-fiction or future of dentistry. Int. J. Oral Sci. 2019, 11, 8. Copyright 2019 Springer Nature.
future of dentistry. Int. J. Oral Sci. 2019, 11, 8. Copyright 2019 Springer Nature.
2.1.1. Physiochemical Synthesis
2.1.1. Physiochemical Synthesis
Biomimetic substitutes developed to replace the natural tooth enamel are synthesized using
Biomimetic substitutes developed to replace the natural tooth enamel are synthesized using
extreme conditions to simulate the natural enamel structure, which includes use of high temperature
extreme
andconditions
pressure to [4].simulate
Chen et theal.
natural
(2005)enamel structure, the
first described which includes
possible use of high of
development temperature
synthetic and
pressure [4]. Chen etnanocrystals,
hydroxyapatite al. (2005) first described
which mimicked thethe
possible
enameldevelopment of synthetic
prism-like structures hydroxyapatite
[4]. Their study
nanocrystals,
showed how which mimickedofthe
a combination enamel
aqueous prism-like and
hydroxyapatite structures
docusate[4]. Their
sodium salt,study
when showed
adjusted tohow a
combination of aqueous
a pH of 5.5, hydroxyapatite
led to precipitation of longand docusate
apatite sodium
crystals aroundsalt, whennm
200–400 adjusted
in size. to a pH of
Further, to 5.5,
limitled to
these rods’
precipitation ofsize,
longthe hydroxyapatite
apatite aqueous 200–400
crystals around solution was
nm replaced
in size. with a fluorapatite
Further, to limit solution and size,
these rods’
processed under extreme hydrothermal pressures, leading to enamel rods with
the hydroxyapatite aqueous solution was replaced with a fluorapatite solution and processed under a size around 5–10
micron
extreme matching the
hydrothermal natural enamel
pressures, leadingsize (Figure rods
to enamel 1a). with
Fan et al. (2009)
a size around developed
5–10 micron controlled
matching
remineralisation of enamel hydroxyapatite crystals using amelogenin and fluoride with a newly
the natural enamel size (Figure 1a). Fan et al. (2009) developed controlled remineralisation of enamel
developed methodology to sequentially form fluoridated needle-like enamel crystals. The biomimetic
hydroxyapatite crystals using amelogenin and fluoride with a newly developed methodology to
solution was prepared using commercially obtained calcium and phosphates at 2.5 mM and 1.5 mM,
sequentially formatfluoridated
respectively, 37 °C with 50needle-like enamel crystals. The biomimetic solution
mM trihydroxymethylaminomethane-hydrochloric acid and was
180prepared
mM
usingNaCl
commercially obtained calcium and phosphates at 2.5 mM and 1.5 mM,
buffer at 7.6 pH. Fluoride concentration from NaF was kept at 1 mg/L to form the desired respectively, at 37 ◦ C
with solution,
50 mM trihydroxymethylaminomethane-hydrochloric
and previously obtained tooth slices were treated with acid3%
andHNO 1803mM NaCl
solution forbuffer at 7.6 pH.
50 seconds
Fluoride concentration
followed by immersion frominNaF was kept atsolution.
the biomimetic 1 mg/LScanning
to form the desired
electron solution,
microscopy and results
(SEM) previously
showed
obtained theslices
tooth formation
wereoftreated
flake-like
withfused
3% crystals on the enamel’s
HNO3 solution surface, which
for 50 seconds followed was byporous in
immersion
structure. CaP (calcium phosphate) nanorods formed were around 25 nm
in the biomimetic solution. Scanning electron microscopy (SEM) results showed the formation in diameter and 100 nm in of
length [5]. A few years later, an approach established by Ren et al. (2012) used a sodium bicarbonate
flake-like fused crystals on the enamel’s surface, which was porous in structure. CaP (calcium
phosphate) nanorods formed were around 25 nm in diameter and 100 nm in length [5]. A few years
later, an approach established by Ren et al. (2012) used a sodium bicarbonate buffer solution and a
mixture containing calcium nitrate tetrahydrate, sodium bicarbonate, disodium hydrogen phosphate,
Biomimetics 2020, 5, 51 4 of 45

and octa calcium phosphate, all maintained at a pH of 6.6, resulting in the formation of human
enamel-like crystals with a size ranging between 100–500 nm [5]. An essential modification applied
to their crystallisation approach was the use of high temperatures (150–200 ◦ C) constantly for 72 h.
However, the pH and pressure range were maintained at standard conditions. More recently, Wang
et al. (2017) described a three-step process to form enamel to mimic the natural enamel formation
sequalae. First, they conjugated the carboxymethyl chitosan (CMC) with a bisphosphonate alendronate,
which stabilised the amorphous calcium phosphate (ACP) to form a CMC/ACP nanoparticle complex.
The second step used a sodium hypochlorite solution to break the CMC/ACP nanoparticles formed in
the first step. Once the nanoparticles were degraded, glycine was added to orient the ACP/CMC to
form a well-organized and spatially arranged enamel rods. This technique is notable as it simulates the
biologic amelogenesis process and supports the initial layering of amelogenin matrix protein, leading
to cell–matrix and matrix–crystal interactions and subsequently to the elongation of apatite crystals [6].

2.1.2. Protein-Matrix-Guided Synthesis

Enamel engineering using protein matrix formation aims to biologically mimic the natural process
of tooth enamel development. It involves the synthesis of amelogenin-rich proteins followed by their
conjunction with calcium and phosphate ions. This technique usually involves enzymatic processing
with enamel proteases like matrix metalloproteases 20 (MMP 20) and kallikrein 4, which provide a
three-dimensional axis for crystal growth [3] (Figure 1b). The three major proteases that pave ways for
enamel matrix development are amelogenin, ameloblastin, and enamelin [7]. During amelogenesis,
each matrix protein plays a vital role in enamel matrix layering and the consequent crystal growth.
Some earlier studies showed the use of octacalcium phosphate solutions along with amelogenin
(10% w/v) to initiate crystal formation [8]. Similar studies using other combinations of enamel matrix
proteins with octacalcium resulted in a more organized, better apatite crystal structure formation [9].
However, these combinations did not completely mimic the natural enamel structure in terms of
their arrangement, hardness, or strength. More recently, studies focussed on understanding how the
amelogenin amino acid chains are formed and the mechanism of their interaction with developing
enamel crystals [10]. For example, Li et al. (2014) described the interaction between a leucine-rich
59 peptide amelogenin synthesized using an alternate splicing technique, which showed a tendency
to form spheroidal nanoparticles arranged in a linear pattern [11]. With the development and
understanding of better biomimetic materials like chitosan, a chitosan gel-based combination of
amelogenin fragments with MMP 20 added to calcium phosphate crystal solution was studied and
tested. This led to the breakdown of amelogenin fragments after primary crystal formation with
improved biomechanical properties for the subsequently formed apatite crystals [12]. However,
even today, most of these methods only manage to focus on matrix formation and crystal formation
as separate entities, and a more sophisticated, novel technique needs to be developed to mimic the
in vivo aspects of enamel formation.

2.1.3. Enamel Surface Mineralisation

As described earlier, enamel mineralisation is a complex and multifactorial process. However,
excessive demineralisation leads to white spot lesions, which marks the initiation of caries. Several
approaches have been tried over the years to induce surface remineralisation. The impacts of fluoridation
on tooth structure has been investigated for over a century now, and the results clearly indicate their
current applications in dentistry (Figure 1c). Fluoride ions usually move the bioavailable calcium and
phosphates from saliva and lead to fluorapatite formation, which has superior resistance to enamel
demineralisation and thereby limits caries progression [13]. Based on this principle, several innovative
oral care products have been developed. Most of them include the use of fluoridated toothpastes
and other fluoride-containing products like varnishes and mouthwashes. Casein phosphopeptide
(CPP)–amorphous calcium phosphate (ACP) combinations are used for improving enamel surface
remineralisation owing to their ability to stabilise calcium, phosphate, and fluoride ions, which are
Biomimetics 2020, 5, 51 5 of 45

already available in saliva, leading to faster and better surface remineralisation of the enamel layer [14].
Ma et al. (2019) conducted a systematic review with metanalyses describing the efficiency of CCP–ACP
in enamel remineralisation. They summarised 12 studies based on the inclusion and exclusion criteria,
which were evaluated either by surface roughness or their microhardness values. The evaluation
showed significant heterogenicity in the surface hardness values, due to which a random model
of analysis was performed which included 5 of the 8 studies selected and which showed values
(SMD = 1.19, 95% CI: [0.72, 1.66], p < 0.00001) indicating that the use of CPP–ACP resulted in superior
remineralisation. The atomic force microscopy (AFM) analysis of three of their studies also showed
that CPP–ACP’s use resulted in reduced roughness of the enamel surface and showed their ability to
repair and form a smooth surface [14,15]. In another study by Fernando et al. which described the use
of SnF2 along with ACP–CCP to induce tooth repair, their in-vitro studies showed the ability of SnF2 to
interact with CPP–ACP complexes to form a nanofilament coating on the tooth surface, with superior
remineralisation activity in comparison to either of these materials individually. The mechanism
involves Sn2 to form cross-links with CPP–ACP to stabilise the bioavailable minerals and to thereby
enhance binding of the ion binding to the tooth minerals. The results showed that this novel combination
can help to significantly improve resistance to caries and dentinal hypersensitivity [15]. A study
by Bossu et al. (2019) compared a biomimetic nanoparticle-infused hydroxyapatite toothpaste with
two other toothpastes with different fluoride concentrations. Their focus was directed towards how
nanoparticle-based HA integrated to enamel surface and formed a coating that is similar to natural
enamel apatite structures. This technique avoids any physiochemical reaction between fluoride ions
and enamel crystals and, at the same time, is more resistant to brushing abrasions and grindings
due to superior chemical bond between the old enamel and new layer of apatite crystals formed [16].
These modifications provide better resistance to caries while prevent the risk of fluorosis due to the
overuse of fluoride-based substituents. Use of bioactive glass for enamel white spot lesions have been
studied extensively in the last few years [17]. These glass particles when in contact with physiological
fluids has the ability to from new apatite crystals, thus essentially remineralising the enamel surface.
Besides, these glasses when incorporated with fluoride formed the more resistant fluorapatite layering
over enamel surface. This has allowed their use in toothpastes, varnishes, and dental cements to treat
carious lesions. In a recent systematic review by Taha et al., they compared the efficiency of different
toothpastes containing fluorides, ACP–CPP combinations and bioactive glass and evaluated the
different studies showing efficiency of each material in improving white spot lesions [18]. Many studies
showed the superior properties of bioactive glasses in forming a mineral layer on an enamel surface
rich in calcium, phosphate, and silica [17]. Some studies showed the improved mechanical properties
in newly formed enamel using bioactive glass-based toothpastes [19,20]. Based on these studies,
bioactive glasses were ranked above both fluoride and casein peptidases in remineralising enamel
white spot lesions and are an effective alternate option.

2.1.4. Cell and Tissue Culture Systems for Enamel Organ Engineering
Enamel tissue engineering approaches ideally include complex interactions between enamel
forming cells (chiefly ameloblasts) with biomimetic scaffolds and enamel proteins to form suitable
in vitro conditions to engineer new enamel crystals (Figure 1d). Although cell- and tissue-based
engineering approaches have been used for developing several organs and structures in the human
body, enamel bioengineering still remains a daunting challenge due to the highly sensitive nature of the
ameloblast cells and the inability to retrieve enamel organ stem cells with superior pluripotency as they
are lost immediately after tooth eruption [21]. The lack of a suitable and more stable ameloblast cell line
is another drawback in enamel tissue engineering. Currently available ameloblast cell lines usually rely
on the feeder layer system to provide sufficient nutritional support or interaction between mesenchymal
cells (feeder layer) to induce primary ameloblast cell growth. The currently available ameloblast cell line
includes the mouse ameloblast-lineage cell line (ALC), the rat dental epithelial cell line (HAT-7), mouse
LS8 cell line, porcine PABSo-E cell line [22], and the rat SF2-24 cell line [23]. The ALC is the oldest of all
Biomimetics 2020, 5, 51 6 of 45

the cell lines and expresses amelogenin and tuftlins that are important markers, indicating their close
relation to ameloblast-like cells. Even the other cell lines mentioned show ameloblastic characteristics,
but each of them moreover focuses on one or other specific markers or areas of enamel formation
and, therefore, still remains an insufficient tool to accurately simulate in vivo enamel development [3].
However, well-characterized, more specifically, human stem cell-derived cell lines in combination with
supporting scaffolds and matrix proteins may help bridge the current knowledge gap and limitations
in synthetic engineering enamel. On successfully developing such biomimetic enamel regenerative
systems, the future of dental restorative therapies can be immensely benefited.

2.2. Biomimetic Aspects of Dentin and Dentin-Pulp-Complex Regeneration

Dentin forms the bulk of the tooth structure and is a highly complex tissue: 70% of the dentin
structure is attributed to its mineral composition, and the remaining 30% is attributed to its organic
content and water. In the tooth structure, there are of two types: (1) peripheral or mantle dentin
and (2) circumpulpal dentin. The mantle dentin forms the hard outer layer of dentin, whereas the
circumpulpal dentin, as its name suggests, makes up the larger part of the dentin and surrounds
the entire pulp tissue [24]. The lower mineral content of the dentin biostructure makes them prone
to quicker demineralisation than enamel. Its complex structure makes its remineralisation slower,
and consequently, the spread of caries into dentin is enhanced, causing infection of the pulp tissue
and, eventually, periapical diseases. In this context, it is essential to develop smart and effective
biomaterials to replace lost tissue mineral and organic content to form dentinal tubules and to induce
remineralisation. Several materials and bioactive analogues can induce this process; however, in this
review, we discuss the key strategies for dentin remineralisation.
Use of resin-based adhesives and bioactive glass for dentin remineralisation is an established
approach for caries affected or partially demineralised dentin, which involves the deposition of
newly formed dentin on the previously carious dentin surfaces [25]. Bioactive glass (BAG) causes
effective remineralisation of dentin and improves the mechanical properties of the dentin structure
through intrafibrillar mineralisation. This allows to maintain the functionality of the tissue in addition
to superior physical properties. However, in this type of dentin formation, lack of seed crystals
or the old dentin crystals will affect the formation of new dentin. For these reasons, ion-based
dentin regeneration may not be sufficient to remineralise dentin that has already been entirely
demineralised by caries [26]. However, ion leaching or an ion releasing approach for dentin caries
removal or remineralisation has been widely used (Table 1). It includes using several bioactive and
biomimetic analogues which not only replace the lost mineral structure but also, at the same time,
protect the collagen fibrils from degradation. The most commonly used biomimetic materials include
bioactive glass (BAG), calcium silicates, calcium orthophosphates, and zinc oxide (ZnO) particles.
Other non-ion-based biomaterials, which are developed more recently and overcomes the drawbacks
of ion-based biomaterials, are described in Table 2.

Table 1. Ion-release-based biomaterials for dentin remineralisation.

Material Modifications and Use Ref.

Modified with zinc, copper, fluoride, and PAA (polyacrylic acid), used as adhesive agents
Bioactive glass (BAG) [27–29]
in dentin resin bonding interfaces, and used in dentin hypersensitivity treatment.
Di/tricalcium silicates and Mineral trioxide aggregate (MTA); bioactivity by alkalisation of
Calcium silicates hydroxyl groups from the CaOH phase, leading to an increase in pH, decreased activity of [30]
MMP, activated mineral precipitation, and antimicrobial activity.
Modifications based on different concentrations of Ca–P, dicalcium phosphate anhydrous
Calcium orthophosphates (DCPA), dicalcium phosphate dihydrate (DCPD), and tetra calcium phosphate (TTCP) as [31]
resin adhesives for dentin remineralisation and improved dentin resin interfaces.
Zinc oxide and Zn-loaded polymeric nanomaterials additives in resin, protective action in
ZnO particles [32,33]
collagen degradation, and initiation of precipitation of poorly crystallised apatite crystals.
Biomimetics 2020, 5, 51 7 of 45

Table 2. Biomimetic analogues for dentin remineralisation.

Biomimetic Analogues Modifications and Use Ref.

Calcium-binding molecule analogous to dentin matrix protein 1(DMP 1) stabilises
Polyacrylic acid9 (PAA9) [34,35]
and controls dimensions of calcium carbonate and calcium phosphate phases.
Functions analogous to collagen-binding matrix phosphoproteins like DMP1 and
Polyvinyl phosphonic acid (PVA) [35,36]
dentin phosphoproteins.
It is a phosphophoryn analogue, binds to collagen fibrils, creates negatively charged
Sodium trimetaphosphate (STMP) [34,37]
sites to receive nanoprecursors, and initiates nucleation of apatite crystals.
Is an analogue for calcium utilisation, released from hardened calcium silicate
Polyaspartic acid (PAS) cements or calcium phosphate mineralising solutions, and assists in controlling the [38,39]
size of ACP nano precursors and their movement into the collagen fibrils.

2.3. Dentin-Pulp Complex Regeneration

When carious lesions are left untreated, they extend beyond hard tissues and cause inflammation
and, in turn, infection of the pulp and surrounding periapical tissues. In these situations, the treatment
plan depends on pulp vitality. In vital pulp therapy, the principle is to induce dentinal bridge formation
to protect the pulp tissue. In cases with a nonvital pulp tissue, till today, root canal treatment is the
standard of care procedure which removes all necrosed or decayed pulp tissue to clean the tooth canals
and to fill them with an inert obturating material which creates a three-dimensional seal both coronally
and apically, thus preventing any microleakage and tooth structure preservation. However, easier said
than done, endodontic treatments are associated with several posttreatment complications, including
periapical pathologies, pain, and secondary infections due to incomplete debris removal or improper
techniques used for pulp therapy [40,41]. A study by Prati et al. (2018) showed that, in a 20-year follow
up study on post-root-canal-treated patients, 15% of subjects developed some form of periapical lesion
and almost 21% of these cases ended in extraction of the tooth based on cumulative teeth survival
statistics [42]. Therefore, it is evident from the previous studies that there are several drawbacks to the
currently used pulp capping and root canal sealing materials, which dictates the need for more refined
biomimetic materials with controlled signalling and a more directed differentiation of the pulp cells to
limit inflammatory responses and to thus allow better healing. Several approaches are currently being
used to fulfil the biologic and structural needs required to eventually preserve the pulp tissue and
tooth structure. These include both cell-free (Table 3) and cell-based therapies (Table 4) for dentin pulp
complex regeneration. Other methods employ a combination of cells and growth factors which are
either directly transplanted to the pulp space (Figure 2) or are laden in combination with biomimetic
scaffolds (Table 5) to induce pulp regeneration.

Table 3. Cell-free therapies for dentin and pulp regeneration.

Biomaterial Indication/Mechanism/Results Ref.

It is a gold standard, with high pH inducing necrosis and mineralisation, good
antibacterial properties, and formation of heterogeneous dentin bridge with tunnel defects;
it increases recruitment, migration, proliferation, and mineralisation of DPSCs and
Calcium hydroxide (CaOH2 ) [43–47]
periodontal ligament stem cells (PDLSCs) through the expression of STRO-1 and CD146
markers; and calcium increases the synthesis of biomolecules such as fibronectin and bone
morphogenetic proteins (BMPs) and causes precipitation mineralisation.
It has an antibacterial effect by releasing calcium hydroxide, a superior sealing ability, low
solubility, higher strength, and more stability than CaOH; it works well in a moist
environment; it forms thicker dentin bridges; it has less inflammatory response,
Mineral trioxide aggregate
hyperaemia and lower pulp tissue necrosis; modifications include calcium chloride
(MTA)/Calcium [45,48–52]
additions, leads to lower setting time, and more biocompatibility; light cured,
silicates/modifications
resin-modified calcium-silicate-based MTA provides immediate polymerization, material
preservation, and superior physical properties; and it induces generation of proangiogenic
factors like IL-8 and IL-beta (interleukins).
Biomimetics 2020, 5, 51 8 of 45

Table 3. Cont.

Biomaterial Indication/Mechanism/Results Ref.

It is a mixture of silica, sodium, and phosphorous oxides with the ability to bond to bone
by controlled release of ions forming apatite crystals repairing hard tissues; it mimics the
natural apatite structure; studies show dentin bridge formation on pulp capping, no
Bioactive glasses [53,54]
necrosis of pulp tissue, and mild inflammatory response; it can form different qualities of
reparative dentin with varying porosities and mechanical properties; and it is noncytotoxic
and improves cell metabolic activities on in vitro testing.
Induces differentiation of DPSCs by MAPK (mitogen-activated protein kinase) and
BiodentineTM calcium calmodulin-dependent protein kinase II (CaMKII) pathways; faster mineralisation [55,56]
of pulp tissue due to the release of transforming growth factor (TGF- Beta 1).
It has dentinogenic, cementogenic, and osteogenic properties; they increase the expression
CEM (Calcium enriched mixture) of fibroblast growth factor 4 (FGF-4) and bone morphogenetic protein 2 (BMP-2), which [57]
favours remineralisation and regeneration.
It has a proliferative effect on pulp tissue comparable to CaOH when used as a lining
material for dentin pulp regeneration, no noticeable antibacterial effect, more
inflammatory response seen on pulp, upregulation of fibroblasts and endothelial cells, and
Glass ionomers and
an inhibitory effect on Hohl cells; in mechanically injured pulp tissue HEMA [58–60]
adhesive resins
(hydroxyethyl methacrylate), it induces secretion of proangiogenic factors like vascular
endothelial growth factors (VEGF) and decreases expression of FGF-2; and concerns
remain regarding the efficiency and quality of tertiary dentin formation after pulp injury.
It is shown to be more effective than CaOH and MTA in differentiation and proliferation of
human tooth germ stem cells; it is highly biocompatible and has known chemotactic effect
Enamel matrix derivatives (EMD) and angiogenic effects; studies indicate their use for periodontal regeneration; and it is [61–63]
inversely shown to cause more inflammation on pulp tissue with little or less hard tissue
formation when compared with CaOH application.
Biomimetics 2020, 5, 51 9 of 46

Figure
Figure 2.
2. Strategies
Strategiesfor
fordentin
dentinpulp
pulpcomplex
complexregeneration:
regeneration: the thecell
cellhoming
homingstrategy
strategyinvolves
involves injection
injection
of
of growth factors and scaffolds into the pulp tissue, which leads to proliferation and migration
growth factors and scaffolds into the pulp tissue, which leads to proliferation and migration of of
progenitor
progenitor cells
cells from
from the
the apical
apical pulp
pulp tissue.
tissue. In
In cell
cell transplantation,
transplantation,the thestem
stemcells
cells are
are injected
injected to
to the
the
pulp
pulp space
space along
along with
with growth
growth factors
factors and
and scaffolds
scaffolds to to induce
induce pulp
pulp regeneration.
regeneration. In In both
both scenarios,
scenarios,
cell
cell growth or proliferation due to growth factors or peripheral induction is followed by
growth or proliferation due to growth factors or peripheral induction is followed by scaffold
scaffold
colonization, which in
colonization, which incases
caseswith
withresorbable
resorbable scaffolds
scaffolds leads
leads to formation
to formation of newof tissue
new tissue over Image
over time. time.
Image reprinted
reprinted with permission
with permission from Morotomi
from Morotomi et al. [64].
et al. [64].

Table 4. Cell-based therapies for dentin pulp complex regeneration (cell transplantation strategies).

Positive Negative
Cells Indications/Mechanism/Result Ref.
Markers Markers
Present next to immature tooth root apex; CD49d,
remains active even in cases of pulp CD51/61,
CD14,
infections or necrosis due to collateral CD56,
Stem cells CD18,
blood supply; has the potential to CD73,
from apical CD34,
Biomimetics 2020, 5, 51 9 of 45

Table 4. Cell-based therapies for dentin pulp complex regeneration (cell transplantation strategies).
Cells Indications/Mechanism/Result Positive Markers Negative Markers Ref.
Present next to immature tooth root apex; remains
active even in cases of pulp infections or necrosis
due to collateral blood supply; has the potential to CD49d, CD51/61, CD56,
Stem cells from apical CD14, CD18, CD34,
differentiate into odontoblast like cells; and shows CD73, CD90, CD105, [65–68]
papilla (SCAP) CD45, CD117, CD150
increased telomerase activity, higher resistance to CD106, CD146, CD166
infection, faster multiplication, and migratory
efficiency within root canals
Derived usually from human third molar pulp
tissue; has high proliferation and colony-forming
ability as dense calcified structures; can CD9, CD10, CD13, CD29,
Dental pulp stem differentiate into osteoblasts, odontoblasts, CD44, CD49d, CD59, CD14, CD31, CD34,
[69–77]
cells (DPSC) adipocytes, and chondrocytes; and can be used as CD73, CD90, CD105, CD45, CD117, CD133
stem cells in neural disorders due to their ability to CD106, CD146, CD166
induce axonal guidance and differentiate into
functional neural cells.
SHED can form bone, dentin, and differentiate into
other nondental mesenchymal cell derivatives; it
Stem cells from human has a higher proliferation rate that DPSC and bone
CD13, CD44, CD73, CD14, CD19, CD34,
exfoliated deciduous marrow-derived MSCs, faster population doubling, [78–82]
CD90, CD105, CD146 CD43, CD45
teeth (SHEDs) and osteoinductive properties; and transplantation
has shown the architecture and cellularity of tissue
formed by SHED to resemble dental pulp closely.
It has ectomesenchyme-derived connective tissue
surrounding enamel and dental papilla; contains
progenitors for cementoblasts, osteoblasts, and
CD9, CD10, CD13, CD29,
PDL; and exhibits the ability to differentiate into
Dental follicle stem CD44, CD49d, CD59, CD31, CD34, CD45,
PDL fibroblasts, to secrete collagen, and to [75,83–87]
cells (DFSC) CD73, CD90, CD105, CD133
consequently interact with fibres in the bone and
CD106, CD166
cementum surface, and DFSC from human third
molars in vitro shows rapid growth and expresses
stem cell markers, including Nestin and Notch 1.

Table 5. Scaffold-based regeneration of the dentin-pulp complex (cell homing strategies).

Scaffold Indications/Mechanism/Results Ref.

It induces apical bleeding, leading to delivery of SCAP to root canal space; it is an autologous
Intracanal blood clot scaffold with growth factors, is clinically efficient, and is economical; and it can be an unstable and [88–91]
unreliable movement of stem cells within the canal space after revascularization.
Autologous injectable scaffold: it can be delivered via collagen sponges; platelet elevation results in
increased production and secretion of growth factors PDGF, TGF-b, Insulin-like growth factor (IGF),
Platelet-rich plasma (PRP) [88,92]
epidermal growth factor (EGF), and epithelial cell growth factor (ECGF), leading to improved
angiogenesis and cell proliferation
Natural polysaccharides from cell walls and seaweeds: stem cells can be incorporated during
scaffold processing; it supports 3-D printing in combination with proteins like DMPs; it includes
Alginate easy diffusion of nutrients and waste debris due to porous structure; it is highly biocompatible, has [93,94]
low immune reactions, is economic, and is easy to fabricate; and has low mechanical strength of the
scaffold when used alone.
Glycosaminoglycans which mimic ECM components: it interacts with stem cell receptors and
drives them towards the area of regeneration; it is shown to have a role in dentin matrix and pulp
Hyaluronic acid (HA) tissue development; it exhibits good biocompatibility, biodegradability, and bioactivity; HA
[90,94–97]
and derivatives derivatives induce proangiogenic factors release; it improves stem cell mineralisation and
odontogenic differentiation; it has low mechanical strength and needs combination with growth
factors to improve regenerative potential; and it may cause hypersensitivity reactions.
Linear amino polysaccharide mimics ECM structure and composition: it is easy to fabricate, is
highly porous, and allows easy migration of cells and growth factors; when fabricated as
nanoparticles, it improved properties due to increased surface area, has better mechanical strength,
Chitosan derivatives [98–100]
and is resistant to enzymatic degradation; it allows the controlled release of growth factors and
improves stem cell or SCAP adhesion, viability, and differentiation; and it is highly biocompatible,
has controlled biodegradation, and has low cytotoxicity with antibacterial properties.
Consists of proteins from hydrolysis of hard and soft tissue-derived collagen; they are
biocompatible and biodegradable, elicits no immune responses, and is cost-efficient; they can be
modified with RBDs (receptor binding motifs), which promotes cell attachment and allows chemical
Gelatin [101,102]
modifications to improve the scaffold’s physiochemical properties; it is used as a drug delivery
medium or in 2D and 3D cultures; and the use of FGF-2 with gelatin shows the formation of
osteo-dentin-like calcified tissue for dentin pulp complex regeneration.
Naturally occurring scaffold obtained from green plants and algae: they are not biodegradable due
to the absence of cellulase enzymes in humans; they possess high tensile strength, high crystallinity,
Cellulose fine fibrous structure, and good formability and is biocompatible; they have higher chances of [103,104]
immune response; and they are used mostly in target-specific drug delivery or growth factor release
in dental tissue engineering.
Biomimetics 2020, 5, 51 10 of 45

Table 5. Cont.

Scaffold Indications/Mechanism/Results Ref.

It is a natural biomaterial, is easily adapted to root canal morphology, and mimics ECM; the most
used is type I, suitable for DPSCs proliferation and mineralisation; it is biocompatible, provides
bioactivity by facilitating adhesion and attachment of stem cells, and induces signalling pathways
Collagen [105–109]
that promote differentiation; the highly porous structure allows easy cell seeding for site-specific
delivery; and commercially available SynnOss (bovine type 1 collagen) in conjunction with
revascularization forms mineralised cementum-like tissues.
Synthetic, biocompatible, biodegradable, nontoxic, 3D matrix gel available as a liquid phase, which
solidifies when in contact with a physiologic salt environment: in vitro studies show pure matrix
Self-assembling peptide
support DPSC cell proliferation and viability when evaluated over three weeks within tooth slices; [110–113]
hydrogels -Puramatrix
puramatrix showed better in vitro results in terms of cell viability and odontogenic differentiation
when used with a co-culture of DPSC/HUVEC (human umbilical vein endothelial cells).
Injectable scaffold with integrated BMP-2, when combined with polylactic acid (PLA) and
polyglycolic acid (PGA), significantly improving the properties and half-life of the PLLA and
prolonged BMP-2 release: it is easily adapts to root canal shape and is biodegradable; it can
Poly L- Lactic acid (PLLA) incorporate drugs/growth factors and is conductive for cells, including DPSC and SHED; it has
[114–116]
nanofibrous microspheres favourable viscosity and porosity; it does not elicit any adverse immune response; it is cheap and
reproducible; the regenerated dentin structure may be disorganized and may not replicate the
natural tooth architecture; and degradation metabolites might cause unfavourable conditions for
surrounding cells but can be excreted to urine without complications.
Poly (lactide-co It has better conductivity for dental pulp fibroblasts proliferation; it is clinically biodegradable, has
gylcolide)-polyethylene glycol fast setting, has low toxicity, has good biocompatibility, and has low immunogenicity; but, it lacks [106,117]
(PLGA-PE) NP intrinsic signalling abilities and is more expensive than other synthetic scaffolds.

Several materials have been developed and tested to evaluate their potential in dentin and pulp
regeneration. So far, a large part of this research focused on comparing these biomaterials in both
in vitro and in vivo studies to develop novel smart biomaterials (Figure 3) [118,119]. These materials
are fabricated in a way that allows easy internal and external modifications based on the inflicted
stimuli and thus fulfils all the biomimetic requirements necessary to initiate tooth hard and soft tissue
regeneration. However, it has been observed that two biomaterials, Mineral trioxide aggregate (MTA)
and CaOH, as well as their modifications were extensively studied in this context by independent
researchers. Tabarsi et al. (2012) [120] studied the effect of MTA and Calcium enriched mixture
(CEM) on rabbit dorsal skin. They used a fresh mixture of both the materials, which was randomly
applied on the skin surface; washed off after 4 h; and evaluated the surface for erythema after 1,
24, 48, and 72 h. The results showed more erythema on the MTA surface as compared to CEM.
On histological examination, MTA showed a higher inflammatory response as compared to CEM.
Their study concluded that CEM is a more biocompatible material for endodontic procedures [120].
A randomized control trial compared CEM and MTA for pulpotomy treatments, which showed no
significant differences in apexogenesis (root closure) radiographically after 12 months follow up [121].
However, Azimi et al. (2014) [122] performed a comparative study between MTA and bioceramic paste,
where no statistically significant result was seen when evaluating the inflammatory response or hard
tissue formation between both the materials after Cvek’s pulpotomy [122]. In one systematic review,
the studies selected compared MTA with CaOH and tricalcium silicate and tested for their success rate,
inflammatory response, and dentin bridge formation. They evaluated 46 studies and showed that MTA
(odds ratio: 2.72) had a significantly higher success rate in all aspects when compared to CaOH. On the
contrary, no noticeable difference was seen between MTA and tricalcium silicates (odds ratio 1.18) [123].
In another systematic review by da Rosa et al. (2018) [124], they compared over 716 papers and
83 patents, which mainly studied CaOH, followed by MTA. The study concluded that MTA surpassed
CaOH in all aspects and was favourable for pulp regeneration [124]. MTA and BiodentineTM were also
compared by Celik et al. (2019) [125] as pulpotomy agents in pulp-exposed carious teeth. Their results
show a 100% radiographic success rate after 24 months for MTA and 89.4% for biodentineTM [125]. It is
evident from these studies that, although the literature has a lot of evidence and data on the biomimetic
properties of each biomaterial discussed here, it is still inconclusive when it comes to individual clinical
implementation. Success in clinical dentin-pulp preservation or regeneration depends on the tooth,
type of carious lesion, and pulp injury.
 MTA and BiodentineTM were also compared by Celik et al. (2019) [125] as pulpotomy agents in pulp-
exposed carious teeth. Their results show a 100% radiographic success rate after 24 months for MTA
and 89.4% for biodentineTM [125]. It is evident from these studies that, although the literature has a
lot of evidence and data on the biomimetic properties of each biomaterial discussed here, it is still
inconclusive
Biomimetics 2020, 5,when
51 it comes to individual clinical implementation. Success in clinical dentin-pulp
11 of 45
preservation or regeneration depends on the tooth, type of carious lesion, and pulp injury.

Figure 3. Smart scaffolds for dentin pulp regeneration. Image adapted from Perez et al. [118] and
Figure 3. Smart scaffolds for dentin pulp regeneration. Image adapted from Perez et al. [118] and
Moussa et al. [119].
Moussa et al. [119].

Although we have developed some highly bioactive and biocompatible materials as medicaments
Although we have developed some highly bioactive and biocompatible materials as
and scaffolds for dentin and pulp repair and regeneration, there is still a need to simultaneously
medicaments and scaffolds for dentin and pulp repair and regeneration, there is still a need to
organize both biological
simultaneously organize and mechanical
both biologicalaspects of these biomaterials.
and mechanical Our
aspects of these primary goal
biomaterials. Ourisprimary
to induce
appropriate
goal is to induce appropriate signalling pathways for cell–cell and cell–matrix interactions while to
signalling pathways for cell–cell and cell–matrix interactions while being noncytotoxic
cells withnoncytotoxic
being superior mechanical properties
to cells with superiorthat will mimic
mechanical in vivo conditions
properties to engineer
that will mimic the dentin pulp
in vivo conditions to
complex.
engineer the dentin pulp complex. Researchers should focus on developing tailor-madewhile
Researchers should focus on developing tailor-made biomimetic analogues keeping
biomimetic
in analogues
mind the essentials of dentin
while keeping in pulp
mind regeneration,
the essentialsincluding revascularization,
of dentin cell, differentiation,
pulp regeneration, including
and growth factor integration
revascularization, with the ability
cell, differentiation, to induce
and growth factorgood qualitywith
integration remineralisation
the ability to of hardgood
induce tissues.
quality remineralisation of hard tissues.
3. Biomimetics in Oral and Maxillofacial Regeneration

3.1. Biomimetics in Bone Regeneration

3.1.1. Bone, a Complex Hub, and a Multitasker

Bone is a highly specialised, complex, and dynamic part of the skeletal system. Apart from the
major function of providing a framework for all the tissues, it is inherently involved in maintaining
several physiological activities, namely haematopoiesis, regulation of ions, maintenance of muscle
mass, and a lot more. It can be classified in two forms: trabecular (medullary) and compact bone
(cortical), both having separate and distinct functions. Trabecular or spongy bone, as its name suggests,
has larger pore size to accommodate hematopoietic cells and comprises the bone marrow, while cortical
bone has more osteocytes and is involved in responding to mechanical signals by bone remodelling
(mechanotransduction) [126–128]. Cortical bone is highly dense (less than 20% porosity) and composed
of closely knit osteons which concentrically form cylindrical systems known as Haversian system,
lodging a blood vessel in the centre (Figure 4). This system has anisotropic mechanical properties, with
the modulus of elasticity (E) = 20 GPa along the Haversian system and E = 8 GPa along the transverse
axis, thus providing a rigid structure [129,130]. On the other hand, cancellous bone has >90% porosity
and is arranged into plates (trabeculae), which offers a larger surface area to mass ratio and better
flexibility with E = 100 MPa [131]. Furthermore, bone comprises organic and inorganic components
(collagen-hydroxyapatite matrix) that provide an interplay of elasticity and rigidity, respectively [132].
Increase in the collagen mineralisation increases the modulus of elasticity, which makes it possible to
bear more stress. In contrast, pure collagen carries the capacity to bear deformation [133]. Furthermore,
bone organization and regeneration by requisition of different molecules like collagen and growth
factors are facilitated by extracellular components like glycosaminoglycans (GAGs) or proteoglycans
(PGs) and by gap junctions [128,134].
 elasticity and rigidity, respectively [132]. Increase in the collagen mineralisation increases the
modulus of elasticity, which makes it possible to bear more stress. In contrast, pure collagen carries
the capacity to bear deformation [133]. Furthermore, bone organization and regeneration by
requisition of different molecules like collagen and growth factors are facilitated by extracellular
components
Biomimetics 2020, 5,like
51 glycosaminoglycans (GAGs) or proteoglycans (PGs) and by gap junctions [128,134].
12 of 45

Figure 4. Macro- and microstructural arrangement of bone: the macroscale structure comprises of
Figure
dense outer4. compact
Macro- and microstructural
bone arrangement
and spongy inner of bone:
cancellous bone. the macroscale
Compact bone structure
is arrangedcomprises of
into osteons
dense outer compact bone and spongy inner cancellous bone. Compact bone is arranged
that form haversian canals. These osteons are formed by fibres arranged in geometrical patterns. into osteons
thatfibres
These form are
haversian
made up canals. These fibrils
of collagen osteons are formed
which by fibres arranged
have alternating in geometrical
organic phases patterns.
to form fibril arrays.
These fibres are made up of collagen fibrils which have alternating organic phases
Each array makes up one collagen fibre. Collagen consists of protein molecules (tropocollagen) formed to form fibril
arrays.
from threeEach array
chains of makes
aminoup one collagen
acids. fibre. Collagen
Image adapted consistsetofal.
from Launey protein
[135]. molecules (tropocollagen)
formed from three chains of amino acids. Image adapted from Launey et al. [135].
Bone has a regenerative capacity of its own and can heal without scarring in case of an uneventful
healing.Bone has a regenerative
However, in cases withcapacity
large orofcritical
its own anddefects,
sized can healit without
requires scarring in support
additional case of an
and
uneventful healing. However, in cases with large or critical sized defects, it requires additional
stabilisation for healing and regeneration. Hard tissue defect’s aetiology in the orofacial region can be
support and stabilisation for healing and regeneration. Hard tissue defect’s aetiology in the orofacial
attributed to genetic or congenital malformations, trauma, infections, cancer, and several other systemic
region can be attributed to genetic or congenital malformations, trauma, infections, cancer, and
or local pathologies. In oral surgery, its application extends from minor defects like periodontal pockets
several other systemic or local pathologies. In oral surgery, its application extends from minor defects
to moderate bone abnormalities like maxillary sinus lift and to much larger bone defects like mandibular
like periodontal pockets to moderate bone abnormalities like maxillary sinus lift and to much larger
andbone
craniofacial reconstruction.
defects like Given
mandibular and the demand,
craniofacial bone regeneration
reconstruction. Given theis of primary
demand, importance
bone in this
regeneration
field and bone is the second most transplanted tissue after blood. Extensive preclinical research
is of primary importance in this field and bone is the second most transplanted tissue after blood. has
been done in this regard, with only a few therapies making it to the clinics [136]. Thus, it is highly
imperative to look for the most efficient and beneficial ways of bone regeneration while ensuring the
integrity and maintenance of the surrounding tissues and its functions. In this section, we will discuss
the ideal requirements for regenerative therapies that will ensure highest biological function of the
regenerated tissue, highlighting the past and current developments in the field.

3.1.2. Determinants of Biomimetics for Bone Regeneration

The prerequisite for biomimetic regenerative therapies is having complete knowledge of the
tissue to be worked with. Given the high complexity and multifunctionality of bone, the standard for
regenerative therapies is set high. The biologically active agents that aid in bone regeneration should
ensure adhesion, migration, proliferation, and differentiation of osteoprogenitor cells, thus resulting in
an acceptable osseointegration. Moreover, considerations for optimal ECM and blood vessel formation
are required for a satisfactory result.
(A) Mechanical and compressive strength: Human cortical bone and cancellous bone compressive
strength ranges from 90–230 MPa and 2–45 MPa, respectively [137]. The compressive and tensile
strength, and the density and fracture toughness of the graft should be comparable to that of the
recipient site. Moreover, it has been shown that the scaffolds’ stiffness can have a direct effect on the
behaviour of the surrounding cells [138]. Therefore, it becomes critical to choose the material according
to the site of procedure and the desired outcome.
(B) Surface properties: Surface differences, even at nanoscale ranges, can affect the behaviour of
cells. They have a regulatory effect over several osteoblastic functions like cell adhesion, migration,
proliferation, cell signalling, genetic expression, and stem cell fate. It is attributed to properties like
increased surface area and roughness; increased wettability and porosity, which in turn increases
nutrient exchange; and protein absorption [139].
(C) Pore size: It is a critical determinant in bone regeneration and repair as it allows for easy
exchange of bone and blood cells, along with other nutrients within the bone. Natural grafts have the
Biomimetics 2020, 5, 51 13 of 45

advantage of inherently possessing the ideal pore size, while several parameters are considered in
fabricating synthetic graft materials. The pore size recommended previously was 0.3 mm to 0.5 mm
to allow proper vascularization and osteogenesis. Pamula et al. used a poly-L-lactide-co-glycolide
(PLG) scaffold with equal pore density but different pore size. Biocompatibility was measured by
comparing the penetration of osteoblast-like cells and the expression of bone reforming proteins like
osteopontin and osteocalcin. The growth and penetration were seen more with pore size 0.4 mm to
0.6 mm, indicating that a larger pore size than recommended before is favourable for osteogenesis [140].
Ghayor et al. demonstrated that material with a pore size of the range 0.7 to 1.2 mm performed better
in in vivo models for calvarial defects [141].
(D) Controlled biodegradability and dimensional stability: Most commonly, the grafts should
be absorbed and replaced by natural tissue over time. Therefore, it requires the material to have
intermediate biodegradability, which corresponds to the simultaneously ongoing natural remodelling
process [142]. If the graft resorbs prematurely, there is a possibility of graft collapse within the
defect and eventually failed restoration. On the other hand, delayed resorption can interfere with
natural bone deposition, elicit immunogenic reactions, and thus decrease biocompatibility. In general,
a decrease in particle size and an increase in porosity of biomaterials reduce the mechanical strength
and enhance the biodegradation rate. Also, nanomaterials undergo faster and more homogenous
biodegradation than conventional micron-based materials. Predictably, biphasic and composite
materials exhibit degradation rates intermediate between the two depending on the percentage of
each phase. For example, Hydroxyapatite (HA)-based materials have a very low degradation rate,
while tri-calcium phosphate (TCP) and organic grafts have higher. Thus, biphasic (HA + TCP) grafts
have to be used with special consideration for the type of recipient site and its degradation profile [143].
Good dimensional stability allows for chairside adaptation of the bone graft to the defect.
(E) Biocompatibility: For a graft to be biocompatible, it should allow growth, differentiation,
and attachment of osteogenic cells and have antimicrobial and appropriate inflammatory properties.
Inflammatory responses to some extent aid in the integration of the graft or implant material as it
leads to remodelling of the tissues by enhancing angiogenesis, by removing debris generated during
the surgical procedure, and by enhancing chemotaxis of reparative cells including pluri-/multipotent
cells [144]. Some materials need to get resorbed by the natural host response mechanisms, while others
which have to be implanted for longer terms require degradation just enough to ensure high tissue
integration. Thus, different levels and extent of inflammatory response are desired depending on
the purpose of the regenerative procedure [145]. Furthermore, in Anthony Gristina’s language,
successful osseointegration is a “race for the surface” between microbial colonization and tissue cell
integration [146]. Harnessing the antimicrobial response is critical to ensure successful graft, further
discussed in the following periodontology section.

3.1.3. Bone Grafts and Scaffolds

(A) Natural grafts
(i) Autograft: It is a part of the patients’ own tissue and has been considered the gold standard for
bone defect repair for years [147,148]. They are commonly obtained from the iliac crest, parietal bone,
and mandible. However, it becomes critical to have a second surgical procedure at the tissue harvest
site, which increases the risk of developing morbid complications such as donor site injury, deformity,
and scarring. It is also associated with higher costs for the surgical procedure, longer recovery times,
excessive bleeding, pain, inflammation, and occasionally infections. However, they have a limited role
in restoration of larger defects, which requires a higher amount of bone. The issues mentioned above
have made other graft materials as a more lucrative alternative in clinical practice [149].
Another exciting autologous bone graft procedure is by ectopic prefabrication, where heterotopic
ossification is achieved to produce bone tissue by placing a scaffold in vivo. It utilises the principle of
Wolf’s law, according to which muscles and bones mechanically and functionally perform together
and have an organizational effect upon one another [150]. Wang et al. successfully demonstrated the
Biomimetics 2020, 5, 51 14 of 45

fabrication of mandibular bony construct by placing a mandible-shaped titanium mesh scaffold packed
with cancellous autologous blocks from dog ribs into the latissimus dorsi muscle with thoracodorsal
artery and vein through the scaffold [151]. Similar studies have been done by Kokemüller et al. (2014),
Naujokat et al. (2019), and Warnke et al. (2004). This method of bone regeneration seems promising,
with an excellent review by Huang et al. [152].
(ii) Allografts include grafts taken from living donors or cadavers and are the second most
commonly used bone grafts [153]. They offer similar advantages as autografts and can be harvested
as various bone matrices: cancellous chips, cortico-cancellous grafts, cortical grafts, osteochondral,
and whole bone segments. However, they have a higher risk of developing immune reactions due to
mismatched genetics. Additionally, there is a risk of transmission of infection. Therefore, the grafts are
devitalised through decalcification, deproteinization, irradiation, and freeze-drying procedures for
preservation. The aforementioned processing makes the osteoinductive potential negligible as the cells
are lost. Blume et al. recently used an allograft to fabricate a customized bone graft using CAD/CAM
technology which fit the large defect perfectly and thus was a clinical success [154].
(iii) Xenografts: Xenografts are any graft across different species. They offer similar advantages
and drawbacks like the allografts, but the immunogenicity is expected to be higher along with
higher risk of cross-species infection. Interestingly, the phenomenon is seen to be lesser with
these grafts, and thus, they have emerged as promising candidates as transplant material [155,156].
Decellularized Bone (DCB) remains the most common graft material. Recently, growth factor-enhanced
DCB has been developed. Also, the addition of doxycycline to Bovine Hydroxyapatite (BHA) was
observed to be more stable than the graft without it, due to its nonantibiotic effects on fibroblasts,
mesenchymal cells, and osteogenic cells, which promote cell adhesion and, ultimately, cell proliferation
and differentiation [157]. Notable FDA-approved DCB products include Graftech® , GraftCage® ,
BTB Select® , BioCAP Select™, MatriGRAFT® , and ReadiGRAFT® [158]. A summary of all the natural
grafts is given in Table 6.

Table 6. Different types of natural bone grafts.

Type of Graft Action Advantage Disadvantage

Donor site injury
Osteogenic Histocompatible
Scarring
Autograft Osteoinductive Negligible immunogenicity
Longer recovery time
Osteoconductive Ideal physical and mechanical properties
Limited size
Histocompatible
Osteogenic Donor site injury
Negligible immunogenicity
Ectopic prefabrication Osteoinductive Scarring
Ideal physical and mechanical properties
Osteoconductive Longer recovery time
No shape or volume limitation
Osteogenic Histocompatible Immune reaction
Allografts
Osteoconductive Ideal physical and mechanical properties. Transmission of infection
Osteogenic Histocompatible Immune reaction
Xenografts
Osteoconductive Ideal physical and mechanical properties. Transmission of infection

(B) Natural polymers.

A diverse group of naturally occurring substances represent the constitution of the extracellular
matrix (ECM). Thus, they can be used as scaffolds for bone regeneration with or without the
combination of stem cells and growth factors, more commonly employed in a technique called guided
bone regeneration (GBR). A few of the polymers, as described by Haugen et al., can be classified as
proteins (collagen, gelatin, and fibrinogen, elastin); polysaccharides (glycosaminoglycans, cellulose,
and amylose); and polynucleotides (DNA and RNA) [136].
(i) Autologous ECM-based substitutes: Comparable performance of decellularized allogeneic
and xenogeneic bone grafts to autogenous bone grafts indicates the importance of ECM in bone.
Paduano et al. examined in vitro osteogenic induction of dental pulp stem cells (DPSCs) by culturing
them on decellularized bone matrix (bECM) as well as collagen type 1 matrix (Col-1). bECM with
Biomimetics 2020, 5, 51 15 of 45

osteogenic growth factors showed maximum expression of osteo-specific markers as compared to the
Col-1 matrix [159].
(ii) Proteins: Collagen, elastin, fibrinogen are some of the abundant extracellular matrix proteins.
Scaffolds derived from these can be manufactured to simulate natural ECM and, when used
with integration of growth factors or living cells, can offer osteoconductive and osteoinductive
properties [160]. More recently, bioinspired proteins have gained attention as biomimetic scaffolds for
hard tissue regeneration [161].
(iii) Marine products: They offer a possible source of bone substitutes as they can mimic bone
with their biochemical composition, structural arrangement, and biofouling ability [162]. Marine
products include collagens from jellyfish, polymers from marine diatoms, chitin from marine sponges,
and hydroxyapatite and calcium phosphates from fishbone and other organisms. [163,164] Sensing
the osteogenic potential of marine products, Pinctada’s powdered nacre was used for maxillary
augmentation by Atlan et al. as early as 1990, while recently, Coringa et al. studied bone substitutes
from oysters in mandibular defects in an animal model [165]. Advances have been made for
chemical modification of marine products and their use as scaffolds for pluripotent cells’ culture [166].
Chitosan, a polymer initially derived from marine organisms, is now widely studied for bone
regeneration [167,168].
(C) Synthetic graft materials
(i) Bioceramics:
Ceramic scaffolds are derived from bioactive inorganic materials. They offer the advantage
of providing a similar biochemical composition of the inorganic phase of natural bone tissue.
Most commonly, Calcium phosphate (CaPs)-derived scaffolds are used, amongst which most popular
are hydroxyapatite (HA) and tricalcium phosphate (TCP). CaPs have the ability for osteoconduction
by allowing osteoblasts to attach, proliferate, and differentiate [169]. On several occasions, CaPs have
been seen to have osteoinductive effects as well [170]. This is attributed to the topography, porosity
(both size and saturation), and composition of these materials, which are believed to allow adsorption,
entrapment, and the final concentration of circulating osteogenic factors and osteoprogenitor cells.
HA and TCP are usually used as biphasic composites (BCP). HA is insoluble and thus maintains the
space and structure, while TCP stimulates new bone formation by the dissolution of calcium and
phosphate ions. Nevins et al. tested different compositions of BCP for alveolar ridge modification,
where similar results were obtained for all, with the only difference being graft resorption. It was
delayed by increasing the amount of HA, which is expected [171]. Janssen et al. used microstructured
TCP in glycerol matrix in cleft palate repair as an alternative to autologous grafts and found them
satisfactory [172]. It can be further highlighted that the origin and method of synthesis of these
scaffolds can have a direct impact on the cellular responses. Marinucci et al. reported higher osteogenic
induction with bovine-derived HA than HA alone when the genetic profiles of mesenchymal stem cells
were compared [173]. Similar comparative studies have been done, which indicate that, even if the
broader outcomes seem similar, there are differences in minute cellular responses which might cause
differences in the regenerative potential and effect of a graft [174]. This variability can be attributed to
the difference in their dissolution/precipitation behaviour, microporosity, physicochemical properties,
surface area, and topography [175]. Thus, the graft materials have to be thoroughly studied and
carefully selected for the intended purpose.
Bioactive glasses that contain calcium can produce a bioactive hydroxy carbonated apatite layer in
biological fluids that can be biologically integrated into the tissues [176]. Further, their resorption rates
can be customized to make it possible to release bioactive molecules at a controlled rate. Although the
brittleness of these materials is a concern, efforts are being made to produce a scaffold of comparable
mechanical properties to that of bone [177].
(ii) Synthetic polymers: For bone regeneration, the most commonly used polymers are aliphatic
polyesters poly-lactic acid (PLA), poly-lactide-co-gylcolide (PGLA), poly-ε-caprolactone (PCL),
polyhydroxyalkanoates (PHA), polyglycerol sebacate (PGS), and poly-glycolic acid (PGA) [178].
Biomimetics 2020, 5, 51 16 of 45

They have tuneable biomechanical, biodegradability, and structural properties. Nevertheless, they
have limited osteoconduction as they have low cell attachment capacity [178]. Also, they are
mechanically stiff, which contradicts the flexibility of natural bone. To overcome the brittleness
of bio-ceramics and stiffness of polymers, composite polymers have been widely explored [179].
Collagen-HA was considered as a promising candidate. Eventually, biomimetic mineralised collagen
(MC) was introduced [180]. Feng et al. showed that MC gave better clinical outcomes for socket
preservation than collagen-HA [181].
(D) Applications and advances
(i) Micro-/nanofibres and nanoparticles
These are produced through electrospinning, which enables the manufacture of extracellular
matrix (ECM)-like structures. They have high porosity, specific surface area, and nano-topography,
modulating cellular behaviour to promote cell adhesion, proliferation, and differentiation by mimicking
the ECM. Several recent studies have reported the differentiation of Mesenchymal stem cells (MSCs) and
other multipotent cells into mineralising osteoblasts when cultured on electrospun nanofibres [182,183].
Electrospun PCL scaffold was used by Puwanun et al. to assess the differentiation of mesenchymal
stem cells from jaw and periosteal tissue [184]. Although clinical studies with nanofibres in dentistry
are still emerging, one of the clinical trials with nanohydroxyapatite was done by Lombardi et al.
Mean sinus pneumatization was significantly lesser (p = 0.15), and crestal bone resorption was less
as well (p = 0.24), indicating the potential role of these therapies in socket preservation after molar,
especially third molar extraction [185].
(ii) Nanocomposites
Nanoscale features have a regulatory effect over profuse osteoblastic cellular functions like cell
adhesion, migration, proliferation, cell signalling, genetic expression, and stem cell fate. Zhang et al.
reported the nanohydroxyapatite’s compressive strength (poly-l-lactic acid nanocomposites), which was
greatly enhanced and reached 115 MPa, comparable to natural bone [186]. Nanohydroxyapatite-covered
polyhydroxy butyrate (PHB) fibres obtained through electrospinning showed better results than
simple PHB scaffolds [187]. Biocompatible nanocomposite with polyurethane, chitosan, and TCA
was fabricated and loaded with amoxicillin, which offers a promising approach for bone tissue
engineering [186,188].
Similarly, drug loading can be enhanced using nanocomposites to elicit drug delivery,
most commonly needed for anti-inflammatory and antibiotic effects. Recently, Shi et al. fabricated
a novel twin-fibre membrane with antibacterial and osteoinductive properties. The drug’s slow
release matched with natural bone regeneration, and the material proved to be biocompatible and
had improved osteogenic properties. It can serve as biomimetic multifunctional artificial periosteum,
the natural layer responsible for bone regeneration [189]. Similarly, a composite scaffold with PLA,
PCL blended with nanoHA, and cefixime complex was synthesized by Sharif et al. with potential
application in oral and maxillofacial related therapies [190].
In conclusion, most widely used bone substitutes in dentistry are still naturally derived grafts and
natural polymers in combination with bio-ceramics. Several recent clinical trials are listed in Table 7.
In a metanalysis study of bone graft substitutes, Corbella et al. found that autogenous bone (AB) alone
leads to significantly higher osteogenesis in comparison to Bovine bone (BB) (p = 0.04) In contrast, no
significant difference was found when BB was compared with a mixture of AB and BB (p = 0.52) [191].
Moreover, BB showed higher bone formation than HA alone (p < 0.001), but a mixture of HA with
TCP showed better results than BB (p < 0.001) [191]. In another metanalysis, Jensen et al. showed that
synthetic bone grafts showed significantly less clinical outcomes [192]. Thus, natural and biologically
closer substitutes offer higher clinical success [192].
Biomimetics 2020, 5, 51 17 of 45

Table 7. Maxillary sinus lift using different bone grafts in oral and maxillofacial surgery.

Ref. Type of Study Type of Graft Method of Evaluation—In Vitro/In Vivo Sample Size Conclusions
MCBA
FDBA
All materials showed good biocompatibility and Osseo
Randomized clinical trial ABB
[193] Histological and histomorphometric analysis 6 patients conductivity with FDBA as the best material, but only one patient
(NCT03496688) EB
per sample was used, so a larger sample size is required.
HA-TCP-30/70
BC
Radiographic analysis, mRNA analysis, Biphasic psychogenic biomaterial (BP) induced a higher
Randomized split-mouth ACB + ABB
[194] histopathological analysis, 8 patients radiographical vertical resorption and graft collapse in comparison
study (NCT03682315) ACB + BP
Immunohistochemistry, TEM with the combination with an organic bovine bone (ABB).
Porcine bone (DPBM) showed comparable results with the widely
11 participants for PPA,
[195] Randomized clinical trial DPBM vs. DBBM CT and trephine biopsy histology used bovine bone (DBBM). A larger sample size and more
12 ITT
extended studies are still required.
Histological BBS remains more stable in terms
MBS
[196] Randomized clinical trial Histomorphometric 60 patients of volume maintenance and radiological graft homogeneity after a
BBS
CBCT healing period of 6 months.
Calcium phosphate crystal double- Histological
Both materials showed comparable histomorphometric and
[197] Randomized clinical trial coated bovine bone and an organic Histomorphometric 33 patients
radiographic results.
bovine bone radiographic
After six months of healing, no statistically significant difference
Randomized split-mouth NHA
[198] Histomorphometric 28 patients was present in histomorphometric outcomes between the NHA
study (NCT03077867) ABB
and ABB groups.
Mineralised solvent-dehydrated bone allograft (MCBA), freeze-dried mineralised bone allograft (FDBA), anorganic bovine bone (ABB), equine-derived bone (EB), synthetic
micro-macroporous biphasic calcium-phosphate block consisting of 70% beta-tricalcium phosphate and 30% hydroxyapatite (HA-TCP 30/70), or bioapatite-collagen (BC); Bio-Oss® Spongiosa
(Autogenous cortical bone (ACB) + Autogenous Bovine Bone (ABB)) Symbios® Biphasic BGM (ACB + Biphasic psychogenic(BP)); Transmission Electron microscopy (TEM); Deproteinized
porcine bone mineral (DPBM), demineralised bovine bone mineral (DBBM), Per-protocol analysis (PPA), Intention to treat analysis (ITT); monophasic bone substitute (100% ß-TCP) (MBS);
a biphasic bone substitute (60% HA and 40% ß-TCP) (BBS); Pure sintered nanohydroxyapatite (NHA); and anorganic bovine bone (ABB).
Biomimetics 2020, 5, 51 18 of 45

3.1.4. Cell Therapy

(A) Stem cells
Cells, either tissue-specific or multipotent, have an immense role in tissue engineering research,
both in vivo and in vitro, and for treatment as well as testing for different grafts/scaffolds. Variations
in the studies call for standardized cell models that can be reproducible in all the settings. Palumbo et
al. compared the behaviour of committed human osteoblast cells from bone biopsies with multipotent
human dental pulp cells (hDPSC) from extracted teeth to identify cellular models for bone regeneration.
They found that committed osteoblast cells are useful for identifying and testing materials and surfaces
osseointegration while hDPSCs are more useful for obtaining in vitro osteocyte-like network for bone
regeneration [199]. Having established the cell models, there have been numerous studies regarding
cell therapies. For bone regeneration, a wide range of pluripotent stem cells have been studied in oral
and maxillofacial surgery, human umbilical cord mesenchymal stem cells [200], bone marrow-derived
stem cells [201], adipose-derived stem cells [202], mesenchymal dental pulp stem cells [203,204],
periodontal ligament stem cells [205], and SHED [206]. While the wide use of these cells seems
promising, strict vigilance and regulation is required to ensure standardized treatment outcomes.
Given the unpredictable nature of multipotent and pluripotent cells, stem cell-derived conditioned
media are getting more attention [199,207,208].
(B) Hybrid scaffolds
CaP-based scaffolds, as discussed above, are osteoinductive due to their nurturing nature towards
the cells and accommodation for growth factors. To potentiate the osteoinductive capacity, scaffolds
integrated with osteoprogenitor cells and factors were developed [204,209,210]. Korn et al. studied
substitutes for autologous grafts in rodent models for alveolar cleft alveoloplasty using bHA alone, bHA
with undifferentiated MSC, and bHA with osteogenically differentiated MSCs, where the last group
showed the best bone growth [211]. Strontium folate (SrFO) derivatives were integrated into biohybrid
scaffolds given their role in treating osteoporosis and other bone diseases. Martin-del-Campo et al.
loaded the SrFO derivatives in TCP and chitosan polyethylene dimethacrylate scaffolds, which were
then seeded with DPSCs. They observed significantly improved results with SrFO-integrated scaffolds
in terms of bone formation [212]. In a clinical study by Al Ahmady et al., autologous bone marrow
mononuclear cells combined with platelet-rich fibrin and nanohydroxyapatite were used to treat
alveolar cleft. It led to lesser donor site complications, faster soft tissue healing, and less postoperative
pain [213].

3.1.5. Cell-Free Therapies

Bone healing is a complex and highly coordinated process, generally divided into inflammation,
renewal, and remodelling (Figure 5). Mimicking this process by using the appropriate growth factors and
small molecules in a regenerative field can help achieve a successful clinical outcome [214]. Furthermore,
these growth factors are also responsible for the recruitment and maturation of osteoprogenitor cells,
especially mesenchymal stem cells. Therefore, they can perform on-site programming of the cells [215].
Biomimetics 2020, 5, 51 19 of 45
Biomimetics 2020, 5, x FOR PEER REVIEW 21 of 46

Figure Thetemporal
Figure 5. The temporalprogression
progression ofof fracture
fracture healing:
healing: healing
healing of a of a fracture
fracture involves
involves a complex
a complex series
series of processes
of processes whichwhich
can becan be broadly
broadly divided divided intophases,
into three four phases, A. inflammatory
A. inflammatory phase;phase;
B. soft B. soft
callus.
callus formation, C. mineralisation of callus, and bone remodelling. Each phase
formation, C. mineralisation of callus, and bone remodelling. Each phase is regulated by key growth is regulated by
key growth
factors, factors,
as shown as shown
in the figure. in the =figure.
CCL2 BMP
CC motif = bone morphogenetic
chemokine called monocyteprotein, FGF = fibroblast
chemoattractant protein,
growth
IL= interleukin, BMP== growth/differentiation
factor, GDF-5 bone morphogenetic protein,factor 5, IGF-1 =angiopoietin
Ang1,2= insulin-like 1growth
and 2, factor
FGF = 1, M-CSF
fibroblast
=growth
macrophage colony-stimulating
factor, GDF-5 = growth/differentiation = osteoprotegerin,
factor, OPG factor PDGF = growth
5, IGF-1 = insulin-like platelet-derived growth=
factor 1, M-CSF
factor, PlGF = colony-stimulating
macrophage placental growth factor, PTH
factor, OPG= parathyroid hormone,
= osteoprotegerin, RANKL
PDGF = receptor activator
= platelet-derived growthof
nuclear factor= κB
factor, PlGF placental SDF-1 =factor,
ligand,growth stromalPTHcell-derived factor
= parathyroid hormone, = transforming
1, TGF-βRANKL = receptorgrowth factor
activator of
β,
nuclear = tumor
TNF-αfactor necrosis
κB ligand, factor
SDF-1 α, and VEGF
= stromal = vascular
cell-derived endothelial
factor 1, TGF-β =growth factor. growth
transforming Image adapted
factor β,
from
TNF-αYague et al.necrosis
= tumor [216]. factor α, and VEGF = vascular endothelial growth factor. Image adapted from
Yague et al. [216].
(A) Bone morphogenetic protein (BMP)
It(A)
has emerged
Bone as the most
morphogenetic promising
protein (BMP) medium for bone regeneration [217,218]. Sudheesh et al.
studied a biphasic
It has emergedpolycaprolactone
as the most promisingconstruct combined
medium withregeneration
for bone hyaluronic acid-based hydrogel et
[217,218]. Sudheesh and
al.
loaded with BMP-2 for correction of vertical bone height in the mandible
studied a biphasic polycaprolactone construct combined with hyaluronic acid-based hydrogel andin rabbits. The outer wall of
the biphasic
loaded with material
BMP-2 for simulated
correction cortical bone,bone
of vertical while the core
height simulated
in the mandible medullary
in rabbits.bone. BMP was
The outer wall
released in a sustained
of the biphasic material manner fromcortical
simulated the construct, while it
bone, while provided
the mechanical
core simulated and space
medullary maintenance
bone. BMP was
properties
released in through the cortical
a sustained mannerpart from
and osteogenesis
the construct, andwhile
angiogenesis through
it provided the medullary
mechanical [219].
and space
Gene delivery is also a lucrative method as it ensures prolonged and stable
maintenance properties through the cortical part and osteogenesis and angiogenesis through the production of the protein,
reviewed
medullaryby[219].Park et al. for
Gene BMP2 is
delivery in also
dentistry [220]. Inmethod
a lucrative a meta-analysis for animal
as it ensures studies
prolonged andforstable
gene
delivery in maxillofacial bone defects, Fliefel et al. found gene delivery as better
production of the protein, reviewed by Park et al. for BMP2 in dentistry [220]. In a meta-analysis for therapeutics [221].
(B) studies
animal The vascular endothelial-derived
for gene growth factor
delivery in maxillofacial bone (VEGF), and IGF1
defects, Fliefel and
et al. 2
found gene delivery as
The VEGF pathway
better therapeutics [221]. is considered critical for angiogenesis, which indirectly affects osteogenesis [222].
VEGF(B) acts synergistically with BMP and other growth factors [223].
The vascular endothelial-derived growth factor (VEGF), and IGF1 and 2 Kim et al. used FGF with BMP2 to
showThe improved
VEGF maxillary
pathway alveolar bone regeneration
is considered [224]. FGF inwhich
critical for angiogenesis, low concentration is seenosteogenesis
indirectly affects to improve
osteogenesis.
[222]. VEGF acts synergistically with BMP and other growth factors [223]. Kim et al. used FGF with
Kitamura et al. tested its clinical potential in an RCT and successfully treated patients with
BMP2 to show improved maxillary alveolar bone regeneration [224]. FGF in low concentration is seen
to improve osteogenesis. Kitamura et al. tested its clinical potential in an RCT and successfully treated
patients with periodontal defects [225]. IGF 1 and 2 have roles in osteoblast differentiation,
Biomimetics 2020, 5, 51 20 of 45

periodontal defects [225]. IGF 1 and 2 have roles in osteoblast differentiation, stimulation of bone matrix
deposition, and collagenous and non-collagenous protein expression and thus was tested for osteogenic
differentiation of MSCs by Reible et al. [226].
(C) Platelet-derived growth factor (PGDF)
PGDF promotes osteogenesis as well as angiogenesis and has been shown to be successful in
periodontal tissue regeneration [227]. In a systematic review by Li et al., BMP was concluded to be less
effective than PDGF [228]. A rich source of growth factors is platelet-derived products like Platelet-rich
plasma (PRP) and Platelet-rich fibrin (PRF). PRP is recommended for faster delivery and PRF for
the stable delivery of factors [229]. Recently, Stumbras et al. performed alveolar ridge preservation
using bone substitutes and autologous platelet concentrate, where they found that plasma-rich growth
factors perform better than the grafts [230]. Concentrated growth factor (CGF) has also emerged as an
excellent tool for oral regenerative medicine [231,232].

3.2. Biomimetics in Mucosal Repair

3.2.1. Oral Mucosa

Oral mucosa is one of the most forgiving tissues in our body, which heals rapidly and without
scarring, usually in smaller defects. It can be attributed to increased vascularity and faster turnover
rates. Oral mucosa is a highly specialised tissue which is divided into alveolar and masticatory mucosa,
depending on the site and function. Their healing becomes challenging in case of large defects where
open wounds remain susceptible to infection and wound contracture. In these conditions, grafts are
required to cover up the wound site to make up for the lost tissue and to act as barriers to ensure
uneventful healing by preventing foreign body entrapment (dead cells and debris) and infection.
Grafts for oral mucosal repair become challenging as it is constantly under wet environment due to
oral fluids and is introduced to load due to mastication and food.

3.2.2. Determinants of Biomimetics

Regenerated tissues are inevitably required to mimic the complex organization of mucosal layers
to have an uneventful and successful wound healing. The desired outcomes for successful biomimicry
are organization into multiple functional layers with ECM organization, maintenance of functionality,
exhibition of volume stability, creation of an epithelial barrier, biocompatibility, lack of toxicity,
and immunological rejection [233,234]. The regenerated tissues are inevitably required to mimic the
complex organization of the mucosal layers to have uneventful and successful wound healing.

3.2.3. Mucosal Grafts

(A) Autologous grafts till today remain the gold standard. Conventional techniques that are
still most widely used include local flaps, distal flaps, and free vascularized flaps from a donor
site. However, their limitation in large defects and donor site morbidity leads to the search for new
regenerative scaffolds and tissues. Cell culturing techniques for autologous cell sheet formation
offer advantages of an autologous graft without the donor site’s involvement. Keratinocyte-based
products are the ex vivo produced oral mucosal equivalent (EVPOME), which are constructed by
cultivation of autogenous keratinocytes [235]. Amemiya et al. have done tremendous work over the
years to develop autologous graft cultures over the human amniotic membrane, with a successful
clinical trial done recently (Figure 6) [236]. Fibroblast-based constructs involve culture of embryonic
fibroblasts or autologous fibroblasts, which are cultures over a collagen matrix. The scaffold resorbs
upon transplantation, leaving the ECM and functional fibroblasts which secrete appropriate growth
factors, allowing faster healing of the tissue [237].
Biomimetics 2020, 5, 51 21 of 45

Biomimetics 2020, 5, 51 23 of 46

Figure 6. (a,b) Auto transplantation procedure for an oral mucosal defect for pleomorphic adenoma
Figure 6. (a,b) Auto transplantation procedure for an oral mucosal defect for pleomorphic adenoma at
at
thethe
timetime of surgery
of surgery and 12 and 12 months
months after surgery,
after surgery, respectively;
respectively; (c–f) morphology
(c–f) morphology and keratinand keratin
expression
expression
patterns patternsmembrane-cultured
of amniotic of amniotic membrane-cultured
oral mucosal cells oral mucosal
and oral mucosa; cells and oral and
haematoxylin mucosa;
eosin
haematoxylin and eosin stained mucosal epithelial cells exhibiting seven differentiated and
stained mucosal epithelial cells exhibiting seven differentiated and stratified layers (c) as compared to stratified
layers (c) as compared
oral mucosa in vivo (d);tokeratins
oral mucosa
(green)inexpressed
vivo (d); keratins (green)mucosal
in the cultured expressed in(e)
cells thevs.
cultured mucosal
oral mucosa (f);
cells
and nuclei stained with propidium iodide (red). Scale bars: (c,e) 100 µm and (d,f) 200 µm. 100
(e) vs. oral mucosa (f); and nuclei stained with propidium iodide (red). Scale bars: (c,e) μm
Images
and (d,f)from
derived 200 μm. Imagesetderived
Amemiya al. [236].from Amemiya et al. [236].

(B) Natural polymers and scaffolds

scaffolds
Human-derived
Human-derived Acellular Dermal
Acellular Dermal Matrices
Matrices (ADM)
(ADM) are are one
one of the more common scaffold
materials which
whichmimic
mimicECM ECMtoto provide
provide a favourable environment
a favourable environmentfor cell
forgrowth and growth
cell growth factor
and growth
exchange [238]. [238].
factor exchange Similar scaffolds
Similar can be
scaffolds cangenerated
be generatedof xenogeneic
of xenogeneic origin
originlike
likeporcine
porcinegraft,
graft, which
showed positive outcomes as a scaffoldscaffold [239]. Collagen and chitin fibres are a few of the other natural
polymers which can be used in various forms, microscopic to nanometric, to form scaffolds for cell
proliferation [240].
proliferation [240].AAcomprehensive
comprehensive review
review of synthetic
of synthetic and natural
and natural graft materials
graft materials has been has been
provided
provided by Toledano
by Toledano et al. [241]. et al. [241].
(C) Synthetic polymers
The most common synthetic polymers used as mentioned mentioned above
above as
as well
well are
arepoly(ε-caprolactone)
poly(ε-caprolactone)
(PCL), poly (glycolic acid) (PGA), poly (lactic acid) (PLA), poly (hydroxyl butyric acid), and poly
(hydroxyl valeric
(hydroxyl valericacid),
acid),allall
of of which
which are are resorbable.
resorbable. TheyThey
offer offer
more more significant
significant advantages
advantages over
over natural
natural ones, as they have longer shelf life, can be manufactured in bulk, and have better
ones, as they have longer shelf life, can be manufactured in bulk, and have better control over physicalcontrol over
physical and chemical properties. However, their stiffness and fragility were seen to increase because
they have been recently used mixed with non-resorbable polymers, (MMA)1-co-(HEMA)1 and (MA)3-
co-(HEA)2. As the synthetic grafts have limited biological interactions, they have to be impregnated
Biomimetics 2020, 5, 51 22 of 45

and chemical properties. However, their stiffness and fragility were seen to increase because they have
been recently used mixed with non-resorbable polymers, (MMA)1 -co-(HEMA)1 and (MA)3 -co-(HEA)2.
As the synthetic grafts have limited biological interactions, they have to be impregnated with bioactive
molecules, which can be growth factors directly (BMPs and VEGF) or other molecules with stimulatory
effects like Si and zinc oxide [242]. Electrospun nanofibres, as mentioned before, offer greater surface
area and favourable porosity in addition to the capacity to carry growth factors as well as drugs such
as antibiotics. They can be used as a dressing in relieving patient discomfort in patients with oral
mucosal defects [243]. The upcoming strategies for mucosal regeneration have outstanding clinical
outcomes, are economically sound, and can be harvested in greater quantities, thus providing reliable
alternatives to conventional autografts.

4. Biomimetics of Periodontal Tissue Engineering and Regeneration

Periodontium is another highly specialised tissue in the oral and maxillofacial region. Common
aetiologies for its loss are periodontitis and loss of tooth due to trauma or caries. Periodontitis is
an inflammatory disease that leads to destruction of the tooth attachment apparatus, consisting of
the gingival tissue, alveolar bone, cementum, and periodontal ligament (PDL). It is also associated
with several systemic diseases such as cardiovascular diseases and rheumatoid arthritis. It is one
of the most widespread infectious diseases and is the leading cause of tooth loss in adults [244].
Periodontal treatment aims to arrest further progression of the disease and to restore tissue integrity.
Scaling and root planing are nonsurgical mechanical approaches to control disease progression [245].
Once tissue destruction has occurred, regenerative treatments are needed. Here, we have highlighted
the key strategies in periodontal tissue regeneration, with a brief view on biomimetic strategies for
oral implantology.

4.1. Periodontal Regeneration

4.1.1. Cell-Based Therapies

Currently, there are several surgical techniques used for periodontal regenerative therapy, including
guided tissue regeneration (GTR), bone graft placement, as well as a variety of different biomaterials
and growth factors [246]. Each technique has its advantages, yet the ability to completely regenerate the
damaged periodontal structures has not been achieved in patients, especially in those with advanced
periodontal defects [247]. In recent years, progress in stem cell biology and tissue engineering has
ushered in stem cell-based approaches in regenerative therapies. Many studies have shown that stem
cells can be used in conjunction with different physical matrices to regenerate periodontal tissues
in vivo [248–250]. Stem cells used for periodontal therapy are usually mesenchymal stem cells (MSCs),
since these cells are capable of differentiating into many tissues (bone, ligament, muscle, etc.) [251].
Also, they have an immunosuppressive capacity that gives them essential features in autologous
and allogenic transplantation [252]. MSCs can be isolated from a variety of sources. Studies using
animal models have shown successful periodontal tissue regeneration with MSCs derived from bone
marrow [253], adipose tissue [254], and PDL [255]. While there is no consensus on which tissue source
provides the most appropriate stem cells, researchers have focused on stem cells obtained from the
oral cavity due to their accessibility as well as their high differentiation and proliferation abilities [256].
Oral stem cells have neural crest origin and thus represent a transient population of embryonic
pluripotent stem cells [257]. Induced pluripotent stem (iPS) cells and embryonic stem cells have also
been proposed for periodontal regeneration [258]. However, there seem to be limitations to this and it
has not been carried out in practice [259]. There is evidence that the cells of PDL tissues are able to form
a complete periodontal attachment apparatus [248]. One study examined the immunomodulatory
properties of PDL stem cells, a type of MSC, and found that PDL stem cells inhibited the proliferation
of activated peripheral blood mononuclear cells (PMBCs) via a partly dependent mechanism through
interferon-γ, which is synthesized by activated PMBCs [258,260]. The disadvantage of PDL stem cells
Biomimetics 2020, 5, 51 23 of 45

is that the cell regenerative potential seems to decrease as a function of the age of the donor [261].
Traditionally, PDL cells were collected from extracted teeth, which presented an issue because of the
limited amount of PDL tissue on extracted teeth [255]. This is because stem cells are required in large
quantities to accomplish clinical trials [254]. However, recent studies have shown that PDL stem cells
can be procured from inflamed tissue within a periodontal defect [262]. This provides an easy and
rapid alternative method to obtain PDL stem cells without extracting teeth.
Cells implanted into periodontal defects in immunocompromised rodents can regenerate
cementum and PDL-like structures and can support periodontal tissue repair [263]. Other similar
studies have demonstrated cementum, new bone, and PDL formation in larger animals like dogs [264]
and pigs [265]. One study evaluated the regeneration of peri-implantitis defects in dogs using
genetically modified PDL stem cells. Bone morphogenetic proteins (BMPs) have great potential in the
regeneration of periodontal tissues [266]. Following the ex vivo transfer of the BMP-2 gene into PDL
stem cells using an adenoviral vector, modified stem cells were implanted into the defects. This resulted
in enhanced new bone formation and re-osseointegration in peri-implantitis defects compared with
direct BMP administration to periodontal lesions [267]. Another finding that demonstrates the PDL
cells’ ability for bone regeneration and maintenance is that they release important humoral factors to
maintain alveolar bone. When PDL stem cells are cultured in an indirect co-culture model, they inhibit
osteoclastic activity of alveolar bone-derived stromal cells, therefore preserving alveolar bone [268].
Biomaterials used in combination with stem cells have the potential to improve the beneficial effects
shown by cell therapy and lead to better control of cell delivery to the target site and to decrease the
number of cells lost. They also play an essential role in the delivery of biological agents that enhance
interaction with the host tissue and improve the cell differentiation process [269]. Because there are
several different biomaterials available (natural biomaterials, ceramic biomaterials, and synthetic
polymers), many animal studies have been conducted using different combinations of biomaterials
and PDL stem cells for periodontal regeneration [270]. In one study, Tsunmanuma et al. showed that
canine PDL cell sheets combined with a mixture of collagen and beta-tricalcium phosphate lead to
improved cementum and PDL fibre formation in a canine 1-wall defect model [271]. Biological agents
also have an effect on PDL cells in periodontal regenerating. For example, insulin-like growth factor-1
(IGF-1) has been shown to increase osteogenic differentiation and produces a cascade of downstream
reactions, playing a pivotal role in cell-based periodontal tissue regeneration [272].
Over the past few years, stem cell-based periodontal therapies have begun testing in clinical
settings [273,274]. In a 2016 RCT study by Chen et al., autologous PDL stem cells from extracted
wisdom teeth were used as an adjuvant to graft materials (bovine-derived bone mineral materials) in
GTR therapy to treat periodontal intrabony defects. While there was a significant decrease in bone
defect, no statistically significant differences were detected between the PDL stem cell group and
the control group [275]. However, the study demonstrated that it is safe to use PDL stem cells in
treating periodontal intrabony defects in humans. Stem cell-based regenerative periodontal therapy is
a promising new field that has the potential to prevent tooth loss, to avoid costly treatments, and to
provide more effective and less invasive treatment options. While there remain many issues that
need to be addressed before stem cell therapy becomes widely available, clinicians should continue to
monitor these technologies’ progression.

4.1.2. Cell-Free Therapies

Alveolar ridge deficiencies can complicate implant placement as well as other prosthodontic
reconstruction. Therefore, to gain adequate alveolar ridge dimensions, many bone regeneration
techniques have been used with conjunctive therapy to optimize bone growth. Specifically, Growth
Factors (GFs) and Platelet-Rich Fibrin (PRF) techniques have been shown to aid in bone healing following
graft placements. GFs are expressed during tissue healing and can promote tissue regeneration when
used in surgical procedures [276]. PRF allows us to obtain GFs directly from the plasma and is therefore
widely used to aid tissue regeneration. It has a positive effect on cell proliferation, migration, adhesion,
Biomimetics 2020, 5, 51 24 of 45

and differentiation. Additionally, Strauss et al. have shown the anti-inflammatory properties of
PRF [277], which support their role in wound healing and bone regeneration. According to a systematic
review by Strauss et al., PRF does have a clinical benefit on ridge preservation and in the early phase
of osseointegration. However, pain and soft tissue healing outcomes remain unclear [277]. Another
systematic review by Dragonas et al. reported some benefit on soft tissue healing and post-op and
swelling [278]. PRF used in conjugation with other bone grafts has shown better clinical outcomes
than when used alone [279–282]. In patients with chronic periodontitis, the use of 1% metformin and
PRF showed more pocket depth reduction and relative attachment level than either group alone [283].
Along with open-flap debridement, PRF has been shown to increase canine periodontitis treatment
outcomes in animal studies [284].
Furthermore, growth factors alone or derived from cells have also given promising results,
given the fact that endocrinal secretions of the cells are responsible for the regenerative effects in
tissues [285]. A sequential application of basic fibroblast growth factors and BMP-2 synergistically
promoted differentiation of periodontal ligament cells, suggesting their potential use for periodontal
regeneration [286]. A meta-analysis, however, suggested that recombinant human BMP2 and PDGF-BB,
in its current concentrations, did not induce a significant effect on tooth extraction socket healing,
sinus augmentation, or reconstruction of alveolar defects. However, 0.3 mg/mL rhPDGF-BB may
promote the healing of sockets [228]. Another meta-analysis showed that 0.3% rhFGF2 and 0.3 mg/mL
rhPDGF-BB showed more periodontal regeneration capacity than other concentrations and control
groups [287]. It was also shown that rhBMP-2 substantially increased bone levels in localised alveolar
ridge augmentation procedures [288].
Apart from the growth factors, other proteins like amelogenin have an eminent role in periodontal
regeneration, in addition to the enamel formation discussed above [289]. Specifically, Emdogain,
a porcine-derived extract of enamel matrix with proteins like amelogenin has gained attention due
to its proliferative effect on cementoblasts [290], osteoblasts [291], endothelial cells [291], gingival
fibroblasts [291,292], and periodontal stem cells [293]; to gingival and periodontal fibre growth and
attachment; and to an anti-inflammatory effect. A systematic review conducted by Esposito et al.
found that adjunctive use of Emdogain regenerates around 1 mm more tissue than techniques like GTR
alone. Moreover, Emdogain is simpler to use and has less complications [294]. No firm conclusion
was drawn in other preclinical [295] and clinical systematic reviews, which can be attributed to the
disparate nature of the studies [296].
Although PRF has been shown to have good tissue healing properties as it can be used like a guided
tissue regeneration membrane, one of its main disadvantages is that it resorbs within seven days [297].
Comparatively, other membranes for periodontal regeneration typically require 4–6 weeks [298].
Overall, PRF and GFs have been shown to have significant clinical therapeutic outcomes when used in
bone tissue regeneration. However, more research is necessary to assess their full clinical benefits and
indications [297].

4.1.3. Guided Tissue Regeneration

Guided Tissue Regeneration (GTR) is one of several procedures that has been developed to
treat periodontitis. The biological principle of this procedure is on the basis that a membrane barrier
can prevent undesirable types of tissue cells from migrating into a wound while simultaneously
allowing desired cell types to repopulate the wound, therefore allowing the regeneration of the
desired type of tissue [299]. GTR membranes are surgically implanted, and there are currently two
types of materials used in these barriers in clinical research applications which are non-resorbable
and resorbable materials [270]. Compared to non-resorbable ones, resorbable barriers are often
favoured as they eliminate the necessity for a second surgical procedure to remove the barrier [300].
Non-resorbable materials used in GTRs include polytetrafluoroethylene (PTFE) or titanium [270],
while resorbable materials are made of synthetic or natural polymers with different combinations
of biomaterials that allow the development of membranes with various structural, chemical,
Biomimetics 2020, 5, 51 25 of 45

and mechanical properties [270]. Common resorbable membranes on the market are based on
either polyesters or tissue-derived collagen, which both have limitations including unpredictable
degradation and weak mechanical properties [270,300]. The GTR membranes need to have the
following characteristics: (1) biocompatibility to prevent inflammatory responses when interacting
with host tissues, (2) a proper degradation profile that matches new tissue formation, (3) proper physical
and mechanical characteristics for in vivo placement, and (4) enough sustained strength to adequately
perform barrier function and circumvent membrane collapse. Considering these requirements, several
research groups have been working to design membranes with predictable rates of degradation,
structures to maintain mechanical properties and bioactive properties, such as calcium-phosphate
based growth factors for bone formation [300]. Over the past decades, many different biomaterials
and their combinations have been made and tested with various levels of success to regenerate
destroyed periodontal tissues treated by GTR [301]. To illustrate, a research was aimed to engineer
and regenerate human long bone tissue by creating scaffolds from nanoparticles to mimic the natural
histological structure of human long bone. They focused on polymer nanoparticle compositions due to
its superior mechanical properties, high durability, and surface bioactivity. The group concluded that
they successfully produced degradable, bioactive, and permeable composite hollow fibre membranes
with a wet phase phase-inversion approach for guided and biomimetic bone tissue engineering [302].
In another attempt to form membrane barrier designs for GTR that mimic naturally occurring biological
processes, a study conducted by Zhang et al. used natural eggshell membrane, a semipermeable
membrane with two unique layers [301]. The results from this study showed that the soluble eggshell
protein with poly lactic-co acid nanofibre (SEP/PLGA) electrospun membrane they made formed an
interconnected porous network with strong mechanical properties. Moreover, biological study results
suggested that SEP/PLGA nanofibres have the potential to improve cell attachment, proliferation,
and spreading. Therefore, the study showed the promising potential of SEP/PLGA nanofibres for
future GTR membrane application [303]. Due to the limitations of current common barrier membranes
used for GTR, many researchers have focused on developing improved barrier membranes, often with
designs that mimic biological processes. Although recent studies have shown promising potential
in the field of GTR, further advancements and research are required before they can be used to help
patients [270].

4.2. Implant BIOMIMETICS

4.2.1. Surface Modification and Alternative Materials for Implant Osseointegration

The most frequently used material in dental implantology is titanium; however, other materials
such as zirconia have recently been introduced to the market [304]. Titanium is widely used due to its
biocompatibility and mechanical properties, which allow for its osseointegration. An implant must be
properly osseointegrated into the jaw to ensure long-term stability, resistance of biomechanical forces,
and proper transfer of forces to the alveolar ridge, which in turn preserves bone [305]. Some of the
drawbacks of titanium implants are its grey colour when placed in an aesthetic region in a patient with a
thin biotype [306] and some rare yet reported titanium allergies [307]. The aesthetic disadvantage gave
rise to the zirconium alternative, a bioinert non-resorbable metal oxide, which also shows comparable
osseointegration [304]. A study comparing the peri-implant crevicular fluid (PICF) surrounding
titanium and zirconium implants found no significant differences in the pro-inflammatory cytokine
or bone metabolism mediators with the exception of leptin [308]. This finding correlates with the
biocompatibility of both materials used in implantology. Zirconium implants are not as predictable as
titanium implants. One study found degradation products of a zirconia-based implant only 29 months
postoperatively [309]. Additional alternatives focusing on aesthetics include using a different system
whereby the titanium implant’s external colouring is pink, which reduces the potential of soft tissues
appearing grey while maintaining the predictability of a titanium implant [306].
Biomimetics 2020, 5, 51 26 of 45

Another way to improve the osseointegration of implants is through surface modification of the
titanium and zirconium. A study compared the osseointegration of zirconium implants with their
surface modified through either blasting, etching, or both methods simultaneously [304]. All three
modifications resulted in good biocompatibility and osseointegration when compared to the reference
zirconium implant. They also had a better attachment to the gingival and bone tissue around the neck
area compared to the reference implant [304]. In addition, surface modification using platelet-rich
plasma has also been investigated. Platelet-rich plasma (PRP) is used to deliver osteogenic and
angiogenic growth factors to speed up bone regeneration and tissue repair [310]. It stimulates the
proliferation and differentiation of mesenchymal cells to osteoblasts to help with bone healing [310].
The PRP used in surgery is prepared from the patient’s blood through centrifugation [311], and the
implant’s surface is later treated with the PRP. A randomized controlled study found that the PRP
application onto the implants’ surface enhanced stability and bone healing [312]. However, a recent
systematic review investigating the use of PRP to aid in bone healing and implant success found that,
while most studies had positive results, the predictability and effect that PRP application onto implants
has on bone regeneration, osseointegration, and implant stability and success remains unclear [313].

4.2.2. Antimicrobial/Anti-Inflammatory Aspects of Oral Implantology

The oral cavity utilises a plaque biofilm to shield itself against microorganisms with its constant
contact with various pathogenic microorganisms. However, plaque biofilm is one of the contributing
factors to caries and other various dental diseases, such as periodontitis. To tackle this issue, various
studies have been investigating antimicrobial agents as a solution [314,315]. Unfortunately, results are
not promising due to the decreased efficiency of antibacterial agents as they are released and degraded
quickly [314,315].
To increase antibacterial agents’ efficiency, many studies have been investigating nanoparticles to
ultimately minimize the development of dental diseases [314,316,317]. Nanoparticles carry unique
physiochemical properties, including high charge density and large surface areas, which allow
them to interact with the negatively charged surface of bacterial cells, preventing bacteria’s cellular
functions [314,316,317]. Thus, these properties allow the nanoparticles to maintain high levels of
antimicrobial activity. The potential of nanoparticles can further be applied to the field of periodontology.
The combination of dimethylaminohexadecyl methacrylate (DMAHDM), 2-methacryloyloxyethyl
phosphorylcholine (MPC), and the nanoparticles of amorphous calcium phosphate (NACP) has been
shown to be highly effective against pathogens associated with periodontitis [314,318–320]. In the
presence of an in vitro subgingival biofilm, nanoparticles with Ag+ , Zn2+ , doxycycline, or synergistic
with chlorhexidine (CHX) have also demonstrated the ability to prevent development of pathogenic
microorganisms [314,318,319,321]. Furthermore, the nanoparticles with Ag present in toothpastes have
been shown to diminish the presence of pathogens associated with periodontic diseases [314,322,323].
Glass and glass-ceramics are viewed as another means of enhancing antibacterial activities in the oral
cavity [324]. Such structures are developed commonly by silica, composed of Si4+ , B3+ , and P3+ , and by
the glass’s heat treatment [324–330]. In addition, these compositions play a crucial role in how practical
their antibacterial activities are. With their unique ion-containing matrices, glass and glass-ceramics
possess bioactive properties, including osteoinductive and osteoconductive abilities, allowing them to
bond with bone tissues [324,331–334]. Glass and glass-ceramics have antibacterial activities as their
surface matrices are integrated with ions, including Ag+ , Ce3+ , Cu+ , Sr2+ , and Zn2+ [324,331,335–338].
The rate at which these ions are released is directly correlated to the glass or glass-ceramic surfaces’
roughness level. The higher the surface roughness of the structure, the higher the rate at which the
ions are released. Fernandes et al. noted that, among these ions, Ag+ is the common ion that is doped
on glass and glass-ceramics studied in recent studies [339]. Hence, these materials play a crucial role
at the site of dental implants as Escherichia coli, Staphylococcus aureus, S. epidermidis, and Pseudomonas
aeruginosa are identified to be present in 90% of all dental implants [339–342]. It has been demonstrated
that 45S5 Bioglass, which contains hydroxycarbonate apatite on its surface, can suppress S. sanguis, S.
Biomimetics 2020, 5, 51 27 of 45

mutans, and Actinomyces viscosus [318,339]. A study by Fernandes et al. has shown that 45S5, which
is a glass or glass-ceramics containing silicon, inhibits the development of E. coli, P. aeruginosa, S.
epidermidis, Moraxella catarrhalis, and Enterococcus faecalis [339]. These examples of the glass structures
indicate a means of delivering a significant antimicrobial activity in the oral cavity.
The use of titanium surface coating has been explored as a means to introduce antibacterial
properties on implant settings as titanium is highly biocompatible and resistant against corrosion.
Although titanium alloy alone is not effective to provide antibacterial properties, its combination
with chitosan allows this co-polymer to prevent biofilm formation and bacterial growth on implant
surfaces [343–345]. Chitosan is a linear polysaccharide that consists of N-acetyl-d-glucosamine
and d-glucosamine units for which position 1 and 4 contain B links. Its unique structure
provides chitosan with a range of properties, including its antibacterial ability, biodegradability,
and cytocompatibility [345–352]. However, its variety of biological properties, especially its
antibacterial properties, depends on the characteristic of chitosan, which includes origin, molar
mass, the degree of acylation, and its condition at production. In addition, chitosan can be produced in
various forms, such as nanoparticles, fibres, gels, membranes, and sponges, but each form has different
effects on chitosan’s biological properties [345,353–356]. A study by D’Almeida et al. (2017) found that
a combination of titanium alloy and non-animal chitosan, which was developed using the coupling
agent triethoxysilylpropyl succinic anhydride (TESPSA), was effective against the growth of E. coli
and S. aureus [345,357]. This combination represents as an ideal coating due to the biocompatibility
of titanium surface and the use of non-animal chitosan, which make the combination as non-allergic
and tolerable for the oral cavity [345]. Overall, nanoparticles, glass-ceramics-based materials, and
chitosan-coated titanium represent effective means to hinder the activity of pathogenic microorganisms
where dental implants are placed. There are various combinations of these materials, and further
research on these materials will help determine the most effective one in clinical settings.

5. Conclusions
In this review, we emphasized the current scenario of biomimetic analogues used in dentistry. It is
evident that intensive research over the years has led to the development of highly innovative, futuristic
biomaterials, and techniques to simulate and replace natural structures in the craniofacial region.
Nevertheless, as a biomimetic consideration, naturally derived or biologically close materials are noted
to have better clinical outcomes with higher chances for clinical translation and patient use. This can
be attributed to the multifarious nature of biological systems, which are an interplay of physiological,
physiochemical, mechanical, and metabolic processes occurring simultaneously. Thus, there is a need for
an interdisciplinary approach integrating medicine, bioengineering, biotechnology, and computational
sciences to advance the current research in dentofacial regeneration. A wide range of in vitro and
animal model studies prove that novel treatments are in the pipeline towards ground-breaking clinical
therapies. We conclude that dentistry has come a long way apropos of regenerative medicine; still,
there are vast avenues to endeavour, seeking inspiration from other facets in biomedical research.

Author Contributions: Conceptualisation, S.D.T.; investigation, A.U. and S.P.; writing—original draft preparation,
A.U., S.P., P.K., H.S., K.T.L., M.T., S.Z., and I.H.; writing—review and editing, A.U., S.P., and S.D.T.; supervision,
S.D.T.; project administration, A.U., S.P., and S.D.T. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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