membranes

Review
3D Printed and Bioprinted Membranes and Scaffolds for the
Periodontal Tissue Regeneration: A Narrative Review
Irina-Georgeta Sufaru 1 , Georgiana Macovei 2, *, Simona Stoleriu 3, *, Maria-Alexandra Martu 1 ,
Ionut Luchian 1 , Diana-Cristala Kappenberg-Nitescu 1 and Sorina Mihaela Solomon 1

1 Department of Periodontology, Grigore T. Popa University of Medicine and Pharmacy, Universitatii Street 16,
700115 Iasi, Romania
2 Department of Oral and Dental Diagnostics, Grigore T. Popa University of Medicine and Pharmacy,
Universitatii Street 16, 700115 Iasi, Romania
3 Department of Cariology and Restorative Dental Therapy, Grigore T. Popa University of Medicine and
Pharmacy, Universitatii Street 16, 700115 Iasi, Romania
* Correspondence: [email protected] (G.M.); [email protected] (S.S.)

Abstract: Numerous technologies and materials were developed with the aim of repairing and recon-
structing the tissue loss in patients with periodontitis. Periodontal guided bone regeneration (GBR)
and guided tissue regeneration (GTR) involves the use of a membrane which prevents epithelial cell
migration, and helps to maintain the space, creating a protected area in which tissue regeneration is
favored. Over the time, manufacturing procedures of such barrier membranes followed important
improvements. Three-dimensional (3D) printing technology has led to major innovations in peri-
odontal regeneration methods, using technologies such as inkjet printing, light-assisted 3D printing
Citation: Sufaru, I.-G.; Macovei, G.;
or micro-extrusion. Besides the 3D printing of monophasic and multi-phasic scaffolds, bioprinting
Stoleriu, S.; Martu, M.-A.; Luchian, I.; and tissue engineering have emerged as innovative technologies which can change the way we see
Kappenberg-Nitescu, D.-C.; Solomon, GTR and GBR.
S.M. 3D Printed and Bioprinted
Membranes and Scaffolds for the Keywords: 3D printing; bioengineering; bioinks; bioprinting; GBR; GTR; scaffolds
Periodontal Tissue Regeneration: A
Narrative Review. Membranes 2022,
12, 902. https://doi.org/10.3390/
membranes12090902 1. Introduction
Academic Editors: Periodontitis is a periodontal tissues inflammatory disease, of multifactorial etiol-
Vladimir-Lucian Ene and Ionela ogy [1–4], in which the host’s immune response to the aggression of periodontopathogenic
Andreea Neacsu bacteria plays a key role [5]. This pathology is characterized by the progressive loss of
periodontal attachment, destruction of the alveolar bone, phenomena that, over time, can
Received: 12 August 2022
lead to tooth loss.
Accepted: 15 September 2022
Periodontal treatment includes the elimination or modulation of the factors which led
Published: 19 September 2022
to the appearance and evolution of periodontitis, as well as the correction, within the limits
Publisher’s Note: MDPI stays neutral of the case and the available technologies, of periodontal soft and hard tissues loss [6,7].
with regard to jurisdictional claims in The reconstruction of these defects is essential for the restoration of masticatory, aesthetic,
published maps and institutional affil- phonation functions and for improving the patients’ quality of life. Therefore, the purpose
iations.
of complete periodontal therapy is to regenerate the entire periodontal system, which
includes bone and cement neo-formation, as well as restoring the attachment of periodontal
fibers [8].
The healing following non-surgical periodontal therapy can only provide periodontal
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
repair, represented by the formation of a long epithelial attachment by migrating epithelial
This article is an open access article
cells [9,10]. Therefore, a complete restoration of periodontal anatomy and functionality
distributed under the terms and cannot be ensured [11]. These aspects can be covered by tissue regeneration, with the mobi-
conditions of the Creative Commons lization and involvement of cellular elements—fibroblasts, osteoblasts and cementoblasts,
Attribution (CC BY) license (https:// as well as the signals needed to direct regenerative processes [12].
creativecommons.org/licenses/by/ Guided tissue and bone regeneration (GTR/GBR) are periodontal surgical interven-
4.0/). tions whose main purpose is to restore the periodontal architecture [13,14]. Primarily, these

Membranes 2022, 12, 902. https://doi.org/10.3390/membranes12090902 https://www.mdpi.com/journal/membranes

Membranes 2022, 12, 902 2 of 21

procedures involve the use of a barrier membrane that may or may not be associated with
bone regeneration materials and whose primary function is to prevent epithelial cell migra-
tion into the bone defect [15,16]. Moreover, the barrier membrane also serves to maintain
the space, creating a protected area in which tissue regeneration is favored. There are
four principles considered necessary for successful regeneration, included in the acronym
PASS: (P) primary closure of the wound to ensure optimal healing; (A) angiogenesis for
adequate vascularization of newly formed tissues, with the supply of oxygen, nutrients,
and pro-healing cells; (S) maintaining space for bone neo-formation, preventing inadequate
epithelial cell proliferation; (S) wound stability to include blood clot formation [17].
In addition to strength and stability, a number of other characteristics of the barrier
membrane can influence the regenerative success. These include pore size, permeability
or its architecture [18]. The pore size of barrier membranes has long been a controversial
issue. Membrane pore size can influence cell adhesion as well as progenitor cell migra-
tion [19]; these processes facilitate not only clot formation but also membrane stabilization,
preventing micro-displacements that could disrupt tissue neoformation [20]. Studies have
shown that excessively large pores make membranes less effective against soft tissue cells
migration and proliferation [21]. To date, an optimal size of barrier membrane porosity has
not been confirmed [22].
Over time, there has been a continuous search for ideal materials for tissue regener-
ation and numerous methods have been developed to stimulate the periodontal tissues
repair [23]. The membranes can be classified, depending on their biodegradation capac-
ity, into resorbable membranes (membranes made of synthetic or natural polymers) and
non-resorbable membranes (metallic membranes; membranes made of synthetic polymers,
polytetrafluoroethylene—PTFE) [24]. Furthermore, another classification includes first gen-
eration (non-resorbable), second generation (resorbable) and third generation (membranes
as a product of tissue engineering) membranes [25].
GTR and GBR techniques have recently benefited from remarkable advances in the
field of three-dimensional (3D) printing, tissue engineering and biofabrication [16,26,27],
creating extensive conditions and possibilities for regenerative therapies.
Moreover, 3D printing technology has led to major innovations in periodontal re-
generation methods, allowing the printing not only of biocompatible membranes and
scaffolds, but also of living cells and supporting components in complex 3D functional
tissues, defined as “bioprinting” [28]. The concept of periodontal tissue engineering has
thus emerged, being defined as ”the use of physical, chemical, biological, and engineering
processes to control and direct the aggregate behavior of cells” [29].
Bioprinting technology is a state-of-the-art tool for rendering biofunctional hierarchi-
cal architecture through 3D printing, with one or more types of living cells embedded.
Bioprinting refers to the printing of components that form a specific tissue, including living
cells embedded in matrix materials, to generate analogous tissue structures [29]. Bioprint-
ing techniques bring a biological functionality to a conventional 3D printed scaffold, as it
mimics a cell-to-cell and cell-to-matrix interaction in construction [30]. The material used in
3D bioprinting involves the usage of living biological cells, hydrogels, chemical factors and
biomolecules, under the name of ”bioink”. A difference must be made regarding the types
of printing material, between ”bioinks” and ”biomaterial inks”, as those two terms do not
assimilate each other. While a bioink is cell-laden, acting as a cell carrier and deliverer
during formulation and bioprinting processing, biomaterial inks are cell-free and can only
be seeded with cells after printing [31].
The aim of this paper is to review the present data in the literature, related to 3D
printing and bioprinting techniques and materials, as well as applications in the form of
membranes and scaffolds in the regeneration of the periodontal apparatus.

2. 3D Printing and Bioprinting Techniques

3D printing is a revolutionary technology also known as additive manufacturing [29].
Figure 1 shows a series of milestones in the chronological evolution of 3D printing and
 print most of its components [40]. The first SLS-based 3D printer was available in 2007
[41].
The first prosthetic limb was printed in 2008 [42] and the first blood vessels were 3D
printed in 2009 [43]. The first 3D printed jaw was made in 2012 [44]. The year 2014 marks
Membranes 2022, 12, 902
the first 3D printing of liver tissue [45] but also the appearance of the first desktop3 3D of 21
printer [46]. Rasperini et al. developed and applied the first 3D-printed scaffold for peri-
odontal repair in 2015 [26]. In 2018, the first commercial 3D-printed full human (skin)
product became available [47] and in 2019, Noor et al. succeeded in generating a perfusa-
bioprinting
ble heart withtechnologies. In 1984,[48].
reduced dimensions Charles Hull pave
The 2020s invented stereolithography
the way (SLA)use
for the personalized for
printing 3D objects from digital
of 3D printing in medicine [49]. data [32]. In 1986 Carl Deckard and Joe Beaman invented
selective laser sintering (SLS) [33].

Figure1.
Figure 1. The
The evolution
evolution of 3D printing technologies.
technologies.

Bioprinting was first demonstrated in 1988 by Klebe with a standard Hewlett-Packard

(HP) inkjet printer to deposit cells by cytowriting technology [34]. Moreover, in 1988, the
first SLA 3D printer was available [35]. In 1989, the fused deposition modeling (FDM) tech-
nique was developed by Scott Crump [36]. In 1999, the first organ used for transplantation
(bladder) was printed [37].
In the year 2000, the first extrusion-based 3D printer (3D-Bioplotter) appeared, and two
years after that, the first kidney was printed by micro-extrusion [38]. In 2003 Wilson and
Boland developed the first inkjet bioprinter by modifying a standard HP inkjet printer [39].
In 2005, the RepRap (Replicating Rapid prototyper) project was founded at the University
of Bath, Somerset, UK, with the aim of creating a low-cost 3D printer that would print most
of its components [40]. The first SLS-based 3D printer was available in 2007 [41].
The first prosthetic limb was printed in 2008 [42] and the first blood vessels were 3D
printed in 2009 [43]. The first 3D printed jaw was made in 2012 [44]. The year 2014 marks
the first 3D printing of liver tissue [45] but also the appearance of the first desktop 3D
printer [46]. Rasperini et al. developed and applied the first 3D-printed scaffold for
periodontal repair in 2015 [26]. In 2018, the first commercial 3D-printed full human (skin)
product became available [47] and in 2019, Noor et al. succeeded in generating a perfusable
heart with reduced dimensions [48]. The 2020s pave the way for the personalized use of
3D printing in medicine [49].
3D printing technology has found numerous applications in dentistry and periodontics,
in particular. 3D printing methods have been investigated in the treatment of gingival
lesions (treatment of gingival recessions, gingivectomies or restoration of the smile design),
as well as regeneration of periodontal tissues: alveolar bone, periodontal ligaments (PDL)
and cement [50,51]. Most commonly used biomaterials in periodontal tissue regeneration
3D assisted are presented in Table 1, along with their main advantages and disadvantages.
Membranes 2022, 12, 902 4 of 21

Table 1. Main biomaterials used in scaffold 3D printing.

Material Advantages Disadvantages

Natural polymers
Biocompatible
Collagen Low mechanical properties
Good cell affinity
Alginate Fast degradation rate
Hydrophilicity
Hyaluronic acid Lack of bioactivity
Antibacterial effect
Chitosan
Synthetic polymers
Highly adjustable physiochemical and
Polycaprolactone (PCL)
mechanical properties Low bioactivity
Polylactic acid (PLA)
Wide range of degradation and Slow degradation rate
Polyglycolic acid (PGA)
resorption kinetics Acidic byproducts
Polyethylene glycol (PEG)
Good repeatability
Poly(lactic-co-glycolic) acid (PLGA)
Not compatible with cell encapsulation
Bioactive Stiffness
Bio-ceramics
Biocompatible Brittleness
Hydroxyapatite (HA)
Osteoconductive Low ductility
β-tricalcium phosphate (β-TCP)
Potential osteoinductive Low flexibility
Bioactive glass
Hydrophilicity Inconsistent cell reactions (variations in
surface quality)

The main 3D printing techniques in GTR and GBR include droplet-based printing,
micro-extrusion and light-assisted printing [28].

2.1. Droplet-Based Printing

Droplet-based 3D printed products are the result of independent and discrete droplets
as basic unit. These techniques can be divided inro inkjet 3D printing and electrohydro-
dynamic jetting. Moreover, inkjet printing can be performed either by continuous inkjet
printing or drop-on-demand printing [43].
Inkjet printers used to be the most frequently approached method in both non-
biological and biological applications [28]. The principle of operation involves the con-
trolled passage of a low-viscosity solution flow, an absolutely necessary condition due
to the excessive force required to release droplets when using solutions at higher viscosi-
ties [52]. The fluid subsequently breaks into very small-volume droplets (1–100 pL) after
passing through the holes in the printhead; these droplets are then distributed under the
influence of pressure pulses in predefined locations, to form three-dimensional structures
after solidification [53].
The development of inkjet printers in biological printing is based on classic, two-
dimensional inkjet printers [28]; in the modified version, the ink, the cartridge and the paper
are replaced with biocompatible materials and the control is performed three-dimensionally,
in the three axes (OX, OY and OZ) [54]. Subsequently, printheads with several hundred
individual nozzles were developed [11].
Continuous inkjet printing is based on a natural phenomenon called Rayleigh-plateau
instability, which exhibits the natural tendency for a stream of liquid to undergo a mor-
phological transformation to a train of discrete drops (Figure 2). On the other hand, drop-
on-demand printer produces a droplet when required; droplet deposition is performed by
displacing the nozzle above the desired location before a droplet is ejected [43]. Modulation
and conditions of drop-on-demand printing can also be done thermally, acoustically or
electromagnetically (Figure 2).
 Continuous inkjet printing is based on a natural phenomenon called Rayleigh-plat-
eau instability, which exhibits the natural tendency for a stream of liquid to undergo a
morphological transformation to a train of discrete drops (Figure 2). On the other hand,
drop-on-demand printer produces a droplet when required; droplet deposition is per-
Membranes 2022, 12, 902 formed by displacing the nozzle above the desired location before a droplet is ejected5 [43].
of 21
Modulation and conditions of drop-on-demand printing can also be done thermally,
acoustically or electromagnetically (Figure 2).

Figure 2. Droplet-based
Figure 2. Droplet-based 3D
3D printing
printing techniques;
techniques; DOD:
DOD: drop-on-demand.
drop-on-demand.

A number of major disadvantages, however, make the use of inkjet printers limited. In
A number of major disadvantages, however, make the use of inkjet printers limited.
the case of thermal inkjet printers there is an increased risk of thermal and mechanical stress;
In the case of thermal inkjet printers there is an increased risk of thermal and mechanical
moreover, low directionality and uneven droplet size, as well as frequent nozzle clogging,
stress; moreover, low directionality and uneven droplet size, as well as frequent nozzle
have been reported [55]. The disadvantages encountered in the case of thermal inkjet
clogging, have been reported [55]. The disadvantages encountered in the case of thermal
printers have tried to be overcome by introducing acoustic inkjet printers or electromagnetic
inkjet printers have tried to be overcome by introducing acoustic inkjet printers or elec-
inkjet printers [56]. The concern in the case of electromagnetic inkjet printers is related to
tromagnetic
the inkjet printers
used frequencies (15–25 [56].
kHz)The concern
which in the case
can induce damageof electromagnetic
to the cell membraneinkjet printers
[57].
is related to the used frequencies (15–25 kHz) which can induce
Moreover, a high pressure is required when using inkjet printers, an aspect damage to the which
cell mem-can
branethe
affect [57].cell viability within the bioink. In order to counteract this particular disadvantage,
Moreover, a highjetting
electrohydrodynamic pressure
avoidsis required when by
such pressures using
usinginkjet printers,
an electric an(Figure
field aspect 2)which
[58].
can affect the cell viability within the bioink. In order to counteract this
Furthermore, electrohydrodynamic jetting is particularly suitable for printing bioink with particular disad-
vantage,
high electrohydrodynamic
cell concentration jetting avoidsratio.
and weight/volume such pressures by using an electric field (Fig-
ure 2)The metallic nozzle is filled with bioink and jetting
[58]. Furthermore, electrohydrodynamic is particularly
a spherical meniscus issuitable
formedfor printing
at the tip of
bioink with high cell concentration and weight/volume ratio
the nozzle due to surface tension. A high voltage is applied between nozzle and substrate
The metallic
and droplets nozzlewhen
are ejected is filled
thewith bioink and
electrostatic a spherical
stresses overcome meniscus is formed
the surface at the
tension undertip
aofsufficiently
the nozzle highdue to surface
voltage tension.
[59]. A high
Different voltage
voltages willis generate
applied between
differentnozzle
types ofand sub-
inkjets
strate and droplets
(micro-dripping, are ejected jetting,
intermittent when the electrostatic
breakdown, stresses
etc.), overcome
but the the surfaceused
most frequently tension
are
under a sufficiently high voltage [59]. Different voltages will generate different
independent, discrete droplets [43]. Also, higher voltages will produce smaller droplets [60]. types of
inkjets (micro-dripping, intermittent jetting, breakdown, etc.), but the most frequently
2.2. Light-Assisted 3D Printing
Light-assisted 3D printing uses laser-assisted, photocuring-based or stereolithography
techniques in order to generate printed and bioprinted products.
Laser-assisted printing involves nozzle-free and non-contacting techniques, which
include laser-induced forward transfer (LIFT), laser-induced forward transfer supported by
an absorption film (AFA-LIFT), matrix-assisted laser evaporation direct writing (MAPLE-
DW), biological processing (BioLP) or laser guidance direct writing (LGDW).
 2.2. Light-Assisted 3D Printing
Light-assisted 3D printing uses laser-assisted, photocuring-based or stereolithogra-
phy techniques in order to generate printed and bioprinted products.
Laser-assisted printing involves nozzle-free and non-contacting techniques, which
Membranes 2022, 12, 902 include laser-induced forward transfer (LIFT), laser-induced forward transfer supported 6 of 21
by an absorption film (AFA-LIFT), matrix-assisted laser evaporation direct writing (MA-
PLE-DW), biological processing (BioLP) or laser guidance direct writing (LGDW).
Generally, a laser-assisted printer is composed of: a pulsed laser source, usually a
Generally, a laser-assisted printer is composed of: a pulsed laser source, usually a
nanosecond laser with ultraviolet wavelength; optics necessary for the beam delivery; a
nanosecond laser with ultraviolet wavelength; optics necessary for the beam delivery;
target in the form of a glass/quartz ribbon coated with the bioink; and a receiving substrate
a target in the form of a glass/quartz ribbon coated with the bioink; and a receiving
coated with biopolymer or cell medium (Figure 3) [31]. The laser beam is directed at the
substrate coated with biopolymer or cell medium (Figure 3) [31]. The laser beam is di-
absorbing layer of the ribbon, generating local evaporation and the formation of high-
rected at the absorbing layer of the ribbon, generating local evaporation and the formation
pressure bubbles, propelling
of high-pressure the cell-containing
bubbles, propelling material towards
the cell-containing materialthe receiving
towards thesubstrate
receiving
[43]. Laser parameters need to be accurately set, in order to control the usual photother-
substrate [43]. Laser parameters need to be accurately set, in order to control the usual
mal, photochemical
photothermal, and photomechanical
photochemical laser effects.
and photomechanical laser effects.

Figure 3. Laser-assisted bioprinting.

Figure 3. Laser-assisted bioprinting.
Laser-assisted techniques allow bioprinting with biomaterials of high cell density and
Laser-assisted
viscosity, at a high techniques
resolution. allow bioprinting instruments,
Being nozzle-free with biomaterials of high
they also avoidcell densityor
clogging
and viscosity, at a high resolution. Being nozzle-free instruments, they
high shear stress problems generated by other techniques [61]. Their main disadvantages also avoid clogging
orinclude
high shear
the high stress
costproblems generated
and the complex by other
printing techniques [61]. Their main disad-
technique.
vantages include the high
Stereolithography cost was
(SLA) and the
the complex printing technique.
first commercially available 3D printing technology.
Stereolithography (SLA) was the first commercially
The basic principle of SLA is given by the photopolymerization availableof3D printing
a highly technol-
crosslinked
ogy. The basic principle of SLA is given by the photopolymerization
viscous polymer or prepolymer under exposure to a light energy source (laser or UV), of a highly cross-to
linked viscous polymer
create layered structures [62].or prepolymer under exposure to a light energy source (laser or
UV), to create layered structures [62].
The SLA printing process involves three main stages: exposure to the light radiation
The platform
source, SLA printing processand
movement, involves
polymerthree main
filling stages:
(Figure 4)exposure
[63]. The to the light radiation
polymerization layers
source, platform
are bonded from movement,
the bottom andup,polymer filling
following the(Figure
exposure4) [63]. Theresin
of the polymerization layers
to light radiation;
are
as bonded
one layer from the bottom up,
is polymerized, thefollowing
platform the exposure
descends for of the resinequal
a distance to light
to radiation;
the thicknessas
one
of one layer and builds the next layer until the printing of the digitized 3D objectofis
layer is polymerized, the platform descends for a distance equal to the thickness
one layer and
completed buildsInthe
[63,64]. nexttolayer
order be useduntil
in the printing of
regenerative the digitized
therapy, 3D object
SLA involves theisuse
com-
of a
pleted
slowly[63,64]. In order to beprepolymer
photopolymerized used in regenerative
formulation,therapy,
loadedSLA involves
with the use of a slowly
cells [64].
photopolymerized
Digital projection prepolymer
printingformulation,
(DLP) is similarloaded
to SLAwithprocedure,
cells [64]. with the difference that,
while in the case of SLA the light beam moves, in the case of DLP it is stationary, which
makes DLP a process with a higher printing speed, but less accurate [63]. DLP uses liquid
photosensitive resins which, under the action of light curing, will form the 3D construction,
layer by layer (Figure 5). DLP printers use a system of micro-mirrors that conduct light to
the projection lens [65].
Selective laser sintering (SLS) uses a high energy laser beam to induce the fusion of
the raw material in powder form, layer by layer. SLS does not require additional material
support during printing because the support is provided by the surrounding powder
(Figure 6) [66]. This particular technique uses a high energy laser which selectively fuses
the powdered material by scanning cross-sections generated from the digital file.
 Membranes
Membranes2022,
2022,12,
12,x902
FOR PEER REVIEW 7 of
7 of2221

Figure 4. Stereolithography schematic principle.

Digital projection printing (DLP) is similar to SLA procedure, with the difference
that, while in the case of SLA the light beam moves, in the case of DLP it is stationary,
which makes DLP a process with a higher printing speed, but less accurate [63]. DLP uses
liquid photosensitive resins which, under the action of light curing, will form the 3D con-
struction, layer by layer (Figure 5). DLP printers use a system of micro-mirrors that con-
duct
Figurelight to the projectionschematic
4. Stereolithography lens [65].
principle.
Figure 4. Stereolithography schematic principle.

Digital projection printing (DLP) is similar to SLA procedure, with the difference
that, while in the case of SLA the light beam moves, in the case of DLP it is stationary,
which makes DLP a process with a higher printing speed, but less accurate [63]. DLP uses
liquid photosensitive resins which, under the action of light curing, will form the 3D con-
struction, layer by layer (Figure 5). DLP printers use a system of micro-mirrors that con-
duct light to the projection lens [65].

Membranes 2022, 12, x FOR PEER REVIEW 8 of 22

Figure 5.
Figure Digital projection
5. Digital projection printing
printing principle.
principle.

Selective laser sintering (SLS) uses a high energy laser beam to induce the fusion of
the raw material in powder form, layer by layer. SLS does not require additional material
support during printing because the support is provided by the surrounding powder (Fig-
ure 6) [66]. This particular technique uses a high energy laser which selectively fuses the
powdered material by scanning cross-sections generated from the digital file.

Figure 5. Digital projection printing principle.

Selective laser sintering (SLS) uses a high energy laser beam to induce the fusion of
the raw material in powder form, layer by layer. SLS does not require additional material
support during printing because the support is provided by the surrounding powder (Fig-
ure 6) [66]. This particular technique uses a high energy laser which selectively fuses the
powdered material by scanning cross-sections generated from the digital file.

Figure 6. Selective
Figure laser
6. Selective sintering
laser principle.
sintering principle.

2.3. Extrusion 3D Printing

Of the various 3D bioprinting technologies, extrusion is the most commonly used in
medical applications [57]. Extrusion 3D printing can be performed under thermal and
non-thermal conditions.
Membranes 2022, 12, 902 8 of 21

2.3. Extrusion 3D Printing

Of the various 3D bioprinting technologies, extrusion is the most commonly used
in medical applications [57]. Extrusion 3D printing can be performed under thermal and
non-thermal conditions.

2.3.1. Thermal Extrusion 3D Printing

Thermal extrusion 3D printing can be done by fused deposition modelling (FDM)
or melt electrowriting (MEW) (Figure 7). Fused deposition modelling (FDM) is based
on layer-by-layer printing of thermoplastic polymers. The polymer is heated over its
melting point and, due to a solid-semisolid transition, is then extruded into filaments
with diameters of 160–700 µm, through a nozzle, according to the predefined design [16].
FDM is characterized by high printing speed; in addition, by adding multiple nozzle
heads, various materials can be printed simultaneously [67]. Due to the fact that FDM
uses high temperatures (140–250 ◦ C), it cannot be used in bioprinting; this technique
Membranes 2022, 12, x FOR PEER REVIEW 9 of 22
can be approached, however, for the realization of multiphase scaffolds of periodontal
regeneration [68–70].

Figure7.7.Thermal
Figure Thermalextrusion
extrusion3D
3Dprinting:
printing: fused
fused deposition
deposition modelling
modelling (right)
(right) and
and melt
melt electrowrit-
electrowrit-
ting (left).
ting (left).

2.3.2.The main disadvantages

Non-Thermal Extrusionof3D
FDM include the low resolution and the surface finish that
Printing
may be poor, following the spread
Non-thermal extrusion techniquesof the allow
material
3Dbefore it has
printing by cooled; it should
extrusion withoutbemelting
noted,
however, that these drawbacks can be overcome by decreasing the nozzle diameter
the material. This printing system can use air pressure, a pressurized piston without [71]. a
Melt electrowriting combines FDM with the electrospinning technique to obtain ex-
valve or a screw to extrude cell-free or cell-loaded biomaterials on a substrate, avoiding
tremely thin filaments (generally 2–30 µm), even nanometric [72], directed in porous
the distortion of temperature-sensitive biomolecules and/or cell death (Figure 8) [16].
constructions with high degree of complexity and precision. MEW involves applying an
Extremely important parameters in this technique are represented by the properties
electric field to continuously pull a molten polymer toward a computer-controlled static or
of the material, the quantity and quality of the used additives. Non-thermal extrusion has
rotating collector plate [73]. The essential parameters in the realization of such construc-
been used mainly for making cell-loaded scaffolds at physiological temperatures [77].
tions are represented by the delivery rate of the polymer, the needle diameter, the collector
speed, the distance from the needle tip to the collector, as well as the applied electrical
voltage [16,74]; by adjusting these variables, fibers can be obtained on a scale very close to
native collagen fibers [75]. Thus, MEW is characterized by a remarkable resolution of the
fibers, with the ability to print 1 cm thick structures, which makes this technique ideal for
scaffolding or cell bioprinting. To date, most research has focused on prints in the same
plane as the substrate/construction plate; it has been shown, however, that constructions
in anatomical forms can be obtained from MEW using biomaterials such as hydrogel or
bioceramics [76].

2.3.2. Non-Thermal Extrusion 3D Printing

Non-thermal extrusion techniques allow 3D printing by extrusion without melting
the material. This printing system can use air pressure, a pressurized piston without a

Figure 8. Non-thermal extrusion 3D printing.

 2.3.2. Non-Thermal Extrusion 3D Printing
Non-thermal extrusion techniques allow 3D printing by extrusion without melting
the material. This printing system can use air pressure, a pressurized piston without a
Membranes 2022, 12, 902 valve or a screw to extrude cell-free or cell-loaded biomaterials on a substrate, avoiding
9 of 21
the distortion of temperature-sensitive biomolecules and/or cell death (Figure 8) [16].
Extremely important parameters in this technique are represented by the properties
of the material, the quantity and quality of the used additives. Non-thermal extrusion has
valve or a screw to extrude cell-free or cell-loaded biomaterials on a substrate, avoiding the
been used mainly for making cell-loaded scaffolds at physiological temperatures [77].
distortion of temperature-sensitive biomolecules and/or cell death (Figure 8) [16].

Figure 8.
Figure Non-thermal extrusion
8. Non-thermal extrusion 3D
3D printing.
printing.

Extremelysignificant
Although important parameters
progress has inbeen
this technique
reported inareextrusion-based
represented by the3D properties
bioprinting,of
the material,
further the quantity
improvements and quality
in bioink of theare
availability used additives.
needed; Non-thermal
hydrogels extrusion
with suitable has
printing
been used mainly for making cell-loaded scaffolds at physiological temperatures [77].
properties have been developed, which can ensure a maintenance of the post-printing 3D
Although significant progress has been reported in extrusion-based 3D bioprinting,
shape without compromising the viability of the obtained product [78].
further improvements in bioink availability are needed; hydrogels with suitable printing
properties have been developed, which can ensure a maintenance of the post-printing 3D
3. Applications of 3D Printing in Periodontology
shape without compromising the viability of the obtained product [78].
3.1. 3D Printed Scaffolds in Periodontal Defects
3. Applications
Monophasicofscaffolds
3D Printing in Periodontology
are characterized by the presence of a single compartment,
3.1. 3D Printed Scaffolds in Periodontal Defects
having the characteristic functions of a barrier membrane: maintaining and stabilizing the
embranes 2022, 12, x FOR PEER REVIEW 10 of 22
bone Monophasic
defect subject to regeneration;
scaffolds ensuringby
are characterized bone proliferation,
the presence without
of a single epithelial inter-
compartment, hav-
ference in bone defect;
ing the characteristic controlofofa the
functions healing
barrier processmaintaining
membrane: in time andand space [79]. Moreover,
stabilizing the bone
these
defectscaffolds
subject tocan be loaded with
regeneration; growth
ensuring bonefactors or cells without
proliferation, to promote bone neo-formation
epithelial interference in
[11]. Obtaining
bone alveolar bone tissue,
defect; control facilitated
of the healing by monophasic
process in time and scaffolds,
space [79].isMoreover,
not sufficient
these scaffolds
for restitutio
canadbeintegrum.
loaded withThus, biphasic
growth scaffolds
factors have
or cells been developed
to promote for the periodon-
bone neo-formation [11]. Obtaining
tal ligaments’
alveolar bone tissue, facilitated by monophasic scaffolds, is notpromote
regeneration. Triphasic scaffolds have emerged in order to sufficientthe
forre-
restitutio ad
generationintegrum.
of cementum, Thus,together
biphasicwith the alveolar
scaffolds have beenbonedeveloped
and periodontal
for the ligaments.
periodontal A ligaments’
schematic regeneration.
structure of printed
Triphasicscaffolds,
scaffoldsalong
have with
emergedtheirinmain
ordercharacteristics,
to promote the are pre-
regeneration of ce-
sented in Figure
mentum, 9. together with the alveolar bone and periodontal ligaments. A schematic structure
of printed scaffolds, along with their main characteristics, are presented in Figure 9.

Schematic
Figure 9.view
Figure 9. Schematic view of monophasic
of monophasic and multiphasic
and multiphasic scaffolds. scaffolds.

Carrel et al. [80] developed an extrusion scaffold, consisting of cylindrical filaments

Carrel et al. [80] developed an extrusion scaffold, consisting of cylindrical filaments
of tricalcium beta-phosphate (β-TCP) and hydroxyapatite (HA), arranged in orthogonal
of tricalcium beta-phosphate (β-TCP) and hydroxyapatite (HA), arranged in orthogonal
layers (OsteoFlux®). It was placed in sheep calvary defects and was compared with bovine
bone (Bio-Oss®) and β-TCP particles. In the first 8 weeks the authors observed a significant
increase in bone growth, but no difference was registered in the total of four months [80].
Another scaffold, consisting of 30% HA, 60% β-TCP and 10% tricalcium alpha-phos-
Membranes 2022, 12, 902 10 of 21

layers (OsteoFlux® ). It was placed in sheep calvary defects and was compared with bovine
bone (Bio-Oss® ) and β-TCP particles. In the first 8 weeks the authors observed a significant
increase in bone growth, but no difference was registered in the total of four months [80].
Another scaffold, consisting of 30% HA, 60% β-TCP and 10% tricalcium alpha-
phosphate (α-TCP), also made by extrusion, in the form of a mesh, with a macroporosity
of 60%, was implanted in sheep sinus [81]. The scaffold was well tolerated, generating a
peripheral bone remodeling in the first 45 days after implantation; at 90 days, the formation
of a peripheral lamellar bone was observed, but after 90 days the scaffold continued to
resorb, leaving an incompletely filled bone defect, with areas of fibrous tissue [81].
Cellular loading of hydrogel scaffolds was also attempted, mainly by electrospinning
techniques; these forms of scaffolds have, however, important limitations related to cell
source and culture [11,82,83]. Improved single-phase scaffolds with growth factors have
also been developed. Cho et al. [84] used extrusion to make a polycaprolactone (PCL) scaf-
fold, loaded with poly (lactic-co-glycolic acid) (PLGA) microspheres with morphogenetic
protein 2 and 7 (BMP-2, BMP-7) and connective tissue growth factor (CTGF); the scaffold
was implanted in vitro on the root surface of human teeth with the removed cementum,
in a cementogenic/osteogenic environment. After 6 weeks, all groups delivered with
growth factor showed surface recovery of the dentin with a layer similar to the newly
formed cement, compared to the control. BMP-2 and BMP-7 showed de novo formation of
significantly thicker tissue layers than all other groups, while CTGF and BMP-7 resulted in
significantly improved integration on the dentin surface [84].
Shim et al. [85] compared 3D printed polycaprolactone (PCL) and tricalcium polycaprolact-
one/β-phosphate (PCL/β-TCP) membranes with a conventional commercial collagen mem-
brane in terms of their ability to facilitate GBR, investigating the mechanical properties in dry
and humid environment. Fibroblasts and pre-osteoblasts were seeded in membranes and
proliferation rates and patterns were analyzed with scanning electron microscopy. Subsequently,
the membranes were placed in alveolar defects in beagle dogs. CT and histological analyzes
at eight weeks postoperatively showed that 3D-printed PCL/β-TCP membranes were more
efficient than 3D-printed PCL and substantially better than conventional collagen membranes
in terms of biocompatibility and bone regeneration [85].
Dubey et al. [86] designed, by infusing a PCL mesh made by MEW, with a hydrogel
loaded with amorphous magnesium phosphate (AMP), a membrane reinforced with highly
adjustable bioactive fibers for GBR. This membrane proved that the presence of PCL
networks manufactured by MEW can delay the degradation of the hydrogel; thus, soft
tissue invasion is prevented. At the same time, a mechanical barrier is generated to allow
slower-migrating progenitor cells to participate in bone regeneration [86].
Hsieh et al. [87] investigated the biological characteristics of human PDL cell spheroids
formed on chitosan and polyvinyl alcohol; PDL cell spheroids were cultured in 3D-printed
polylactic acid scaffolds by FDM to assess mineralization capacity. Cellular spheroids
formed on the chitosan membrane demonstrated an increased alkaline phosphatase activity,
as well as an increase in mineralized matrix deposits [87].
Bai et al. [88] have developed an individualized titanium mesh (Ti-Mesh) using
computer-aided design and sintering additive manufacturing technology to evaluate the
effect of different thicknesses and sizes of titanium mesh pores on its mechanical properties.
The authors observed that, as the mesh diameter increased (3 mm to 5 mm), the mechanical
properties of the mesh decreased. The 0.4 mm thick titanium mesh proved to be strong
enough with little mucosal stimulation.
Porous networks of 3D printed titanium-niobium alloy (Ti-Nb) have also been de-
veloped in order to maintain the space, to prevent the fibroblasts’ growth and inhibit
the bacterial colonization [89]. In this technique, Ti-Nb alloy meshes were prepared by
selective laser melting (SLM) and used as substrates for new surface coatings. Porous
coatings of chitosan (CS)/gelatin (G)/doxycycline (Dox) were formed on the meshes, using
electrophoretic deposition (EPD) and lyophilization. This membrane has been shown to
Membranes 2022, 12, 902 11 of 21

be effective in preventing the growth of fibroblasts, while allowing nutrient infiltration;

moreover, its antibacterial effect was observed.
A scaffold with two compartments was designed: bone and ligament, by combining
extrusion with casting; the wax forms were made by extrusion, and the materials were
then cast into these shapes [90]. The bone compartment was seeded with Ad-CMV-BMP7
transduced periodontal ligament cells and the ligament compartment, composed of three
superimposed cylinders, was seeded with PDL cells. The authors observed that parallel
and oblique oriented fibers were generated, which grew and traversed the designed PCL
and poly (glycolic acid) (PGA) structures, forming ligaments and bone structures similar
to the native ones; the disadvantage, however, is the inability to control and predict cell
directionality in vivo [90].
Vaquette et al. designed a scaffold with a bone compartment made by β-TCP FDM,
seeded with osteoblasts, and a ligament compartment produced by electrospinning, in
which PDL cell sheets were placed [69]. Following the in vitro test, it was observed
that osteoblasts formed a mineralized matrix in the bone compartment after 21 days
of culture and that harvesting the PDL cell sheet did not induce significant cell death. The
biphasic scaffold seeded with cells was placed on a block of dentin and implanted for
8 weeks in a subcutaneous model of athymic rat. The authors observed bone neoformation,
ligament and cement regeneration (but with perpendicular non-orientated non-functional
periodontal fibers) [69]. This model was taken over and modified by Costa et al. [91];
the bone compartment, with an increased pore size, was covered with a layer of calcium
phosphate (CaP) to increase osteoconductivity, seeded with osteoblasts and cultured in vitro
for 6 weeks. Then PDL cell sheets were placed in the obtained product, subsequently
implanted subcutaneously in athymic rats for 8 weeks. These modifications led to better
bone neo-formation, better oblique orientation of periodontal fibers (but poorly controlled)
and increased vascularization [91].
Wang et al. [92] developed a biphasic scaffold model made of collagen and strontium-
doped calcium silicate, manufactured by extrusion, loaded with gingival fibroblast cells; it
was placed in calvary defects in rabbits. The authors found that biphasic scaffolds improved
osteogenesis [92].
Lee et al. developed a triphasic scaffold with a precise architecture, enriched with bio-
chemical gradients [70]. This scaffold was made up of three distinct, layered compartments,
corresponding to the morphology of the periodontal complex: cementum, periodontal
ligament and alveolar bone. Each component had a specific architecture, with variable
pore sizes (100, 600 and 300 µm). Moreover, PGA microspheres loaded with growth factors
specific to the regeneration of each tissue type (amelogenin, CTGF and BMP-2) were added
to each compartment. The scaffold was digitally designed and 3D printed, but the addition
of microspheres was done manually, by pipetting. The authors observed a discontinuous
cementogenesis, with a notable osteogenesis in the bone compartment. Interposed connec-
tive tissue between these two mineralized formations was found, with an alignment of the
fibers and a ligament attachment on the newly formed cementoid tissue [70].
Rasperini et al. [26] designed a customized scaffold, based on the patient’s computed
tomography; the scaffold was made of PCL powder containing HA, using selective laser
sintering. Although this scaffold proved effective in treating large periodontal defects, at
12 months it became exposed, which compromised the therapeutic result.
Therefore, triphasic scaffolds, with a three-compartment design, have an important
potential in complete periodontal regeneration, which involves the formation of cement
on the root surface, the formation and proper insertion of periodontal fibers in hard
tissues and sufficient rigidity in general [93], but their design and implementation require
further investigation.
One concern in making and functionalizing multiphase scaffolds was poor interphase
cohesion, which affects the mechanical stability of the scaffold. Continuous additive
fabrication or simultaneous multiphase crosslinking seem viable options to overcome this
dilemma [94,95].
Membranes 2022, 12, 902 12 of 21

3.2. Socket Preservation

Bone resorption after dental extraction represents a common challenge and various
techniques have been developed and investigated, 3D printing included, in order to pre-
serve an adequate height of the alveolar bone. Park et al. [96] evaluated the efficiency of
a 3D printed PCL scaffold implanted in artificially created saddle-type bone defects in
beagle dogs, along with β-TCP. The authors observed that the PCL scaffold maintained the
physical space, without any inflammatory infiltrates around the scaffold.
Goh et al. [97] inserted a 3D bioresorbable PCL scaffold in fresh extraction sockets
in a randomized controlled clinical trial (RCT). The micro-CT and histological analysis
revealed that this treatment option resulted in a lower vertical ridge resorption at the
6-months evaluation.
3D printing was used also for HA granules manufacturing, which were placed in
alveolar sockets after atraumatic extractions and covered with collagen membrane [98].
At the 8-weeks evaluation, the grafted site was completely filled with woven bone, with
vascular neo-formation and without any inflammatory signs.

3.3. Other Applications

Positive outcomes have been observed in bone augmentation for sinus lift procedures
when using 3D applications [99]. Different materials have been tested, such as monolithic
monetite (dicalcium phosphate anhydrous) or biphasic calcium phosphate [100]. The main
advantages of using 3D printed products for sinus lift are represented by the low risk of
infection transfer, as well as by shorter surgery time and higher comfort for the patient [101].
A synthesis of the main 3D applications in available studies is presented in Table 2.

Table 2. Applications of 3D printing in periodontology.

Application Authors Type of Study Method Material 3D Printer

Poly-epsilon
Kim et al., 3D-printed tooth
In vivo caprolactone and Not mentioned
2010 [52] scaffold
hydroxyapatite
3D wax-printing system
Park et al.,
In vivo 3D-printed scaffold PCL-PGA (ModelMaker II, Solidscape, Inc.,
2010 [90]
Merrimack, NH, USA)
Carlo Reis et al., PLGA/CaP bilayered
In vivo 3D-printed scaffold Not mentioned
2011 [102] biomaterial
3-D rapid prototyping wax printer
Park et al., Poly-ε caprolactone
In vivo 3D-printed scaffold (ModelMaker II; Solidscape Inc.,
2012 [68] solution (PCL)
Merrimack, NH, USA)
Obregon et al.,
In vivo 3D-printed scaffold Bilayered biomaterial Not mentioned
2015 [103]
Vaquette et al., FDM + solution FDM, Osteopore Inc. Singapore
In vivo PCL
2012 [69] electrospinning In-house solution spinning device
Costa et al.,
In vivo 3D-printed scaffold Bilayered biomaterial Not mentioned
GTR 2014 [91]
Park et al.,
In vivo 3D-printed scaffold Gelatin, chitosan Not mentioned
2014 [104]
Lee et al., Layer-by-layer PCL +
In vivo Bioplotter, EnvisionTEC
2014 [70] deposition hydroxyapatite
3D-printed SLS (Formiga P100 System; EOS
Rasperini et al.,
Case report Bioresorbable PCL e-Manufacturing Solutions,
2015 [26]
Scaffold Pflugerville, TX, USA))
Sumida et al.,
RCT 3D-printed scaffold Titanium Not mentioned
2015 [105]
Pilipchuk et al.,
Preclinical study 3D-printed scaffold PCL Not mentioned
2016 [106]
Adel-Khattab R1Series ExOne (PROMETAL,
In vitro 3D-printed scaffold Bioceramic
et al., 2018 [107] North Huntingdon, PA, USA)
Lei et al. 3D-printed bone
Case report Not mentioned Not mentioned
2019 [108] model
Bartnikowski Layer-by-layer Bioplotter, EnvisionTEC, Dearborn,
RCT PCL
et al., 2020 [109] deposition MI, USA
Membranes 2022, 12, 902 13 of 21

Table 2. Cont.

Application Authors Type of Study Method Material 3D Printer

3D-printed
Goh et al., FDM techniques (FDM 3000;
Pilot RCT bioresorbable PCL
2015 [97] Stratasys, Eden Prairie, MN, USA)
scaffold
Kijartorn et al., Hydroxyapatite
Socket Prospective study 3D-printed scaffold Projet 160, 3D systems
2017 [98] granules
preservation 3D bioprinting system (laboratory
3D-printed
Park et al., -made system in Korea Institute of
In vivo bioresorbable PCL
2018 [96] Machinery and Materials, Daejeon,
scaffold
Korea)
3D-printed 3D-powder
Tamimi et al., Synthetic calcium
Case report monolithic Printing system (Z-Corporation,
2009 [110] phosphates
Vertical bone monetite blocks Burlington, MA, USA)
augmentation 3D-printed 3D-powder
Torres et al., A/b-tricalcium
In vivo monolithic Printing system (Z-Corporation,
2011 [100] phosphate
monetite blocks Burlington, MA, USA)
Sinus Mangano et al., 3D synthetic bone
In vivo Ceramic Not mentioned
augmentation 2015 [103] substitute
Di Giacomo et al., SLA surgical Simplant CSI Materialise, Ann
NRCT Polymer
2005 [111] guides Arbor, MI, USA
SLA surgical guide (External Hex
Cassetta et al., 3D-printed surgical
Retrospective Acrylic Safe1, Materialise Dental, Leuven,
2013 [112] guide
Belgium)
Pozzi et al., SLA surgical Nobel Procera, Nobel Biocare,
Clinical trial Acrylic resin
Guided 2014 [113] guides Zurich, Switzerland
implant Stübinger et al., 3D-printed surgical
Prospective Polymer Astra Tech AB, Mölndal, Sweden
placement 2014 [114] guide
Shen et al., Geomagic, version 10.0, Geomagic,
RCT SLA templates Acrylic
2015 [115] Research triangle Park, NC, USA
Verhamme et al., 3D-printed surgical NobelGuide (Nobel Biocare,
Prospective Not mentioned
2015 [116] guide Gothenburg, Sweden
Xu et al., SLA surgical
In vitro Acrylic Conne×350; Objet, Rehovot, Israel
2016 [117] guides
Bernard et al., SLA surgical Simplant; Materialise Dental,
RCT Acrylic
2019 [118] guides Waltham, MA, USA

Other applications include 3D-surgical guide precise implant placement, with both
static and dynamic implantation guiding [111–118]. 3D printing has been also extensively
used in education of dental school students and residents and even for patient motivation.

4. Bioprinting
The 3D bioprinting process comprises six major stages:
a. Data acquisition, using X-ray scanning and reconstruction techniques, computed
tomography (CT), magnetic resonance imaging (MRI), or directly using computer-
aided design (CAD) software. These data will be processed with the help of specific
software. The file is converted to a printer-readable file [119]. The data is then
translated to allow estimation of the amount of material to be extruded, which
depends on the desired height and width of the layer according to the shape of the
bioink (droplet or filament) [31,120].
b. The choice of bioink, which is made according to the printing technique and the
requirements of the printed structures. Thus, the bioink must meet favorable mechan-
ical properties, as well as biocompatibility and printability requirements. The bioink
can contain isolated cells, growth factors and bioprinting materials. It is prepared
according to the physiological temperature, pH and requirements of the printed
structures [31].
c. Setting the appropriate printing parameters, depending on the bioink and the desired
structure of the printed product.
Membranes 2022, 12, 902 14 of 21

d. The actual bioprinting, under close observation to make adjustments when necessary.
Printing resolution is specific to the printer and the type of bioink. In cases of high
resolution, the time to fabricate the object can be longer [121].
e. Post-printing stage, which can include spinning and microscopical assessment of the
printed object. The bioprinted object is kept in an incubator or bioreactor.
f. Placement of the bioprinted product (in vivo or in vitro conditions).
3D bioprinting involves precise, layer-by-layer positioning of biological materials
and living cells [57]. Some applications of 3D bioprinting include stem cell research [122],
anti-cancer therapy [123], drug testing [124] and tissue engineering [125]. In tissue engi-
neering, these technologies can control pore size, shape, distribution and interconnectivity.
Moreover, by associating bioprinting technologies with imaging tests, such as cone-beam
computed tomography (CBCT), specialized constructions can be made, adapted to the case
and site-specific [126].
Bioprinting is based on three fundamental concepts: biomimicry, self-assembly and
building blocks with mini-tissue [127]. Biomimicry involves the creation of exact replicas
of the cellular and extracellular parts of a tissue and organ [128]. So far, bioprinting has
been adopted for the manufacturing of bioartificial tissues and organs, such as skin, bones,
cartilage, kidneys, heart, lungs [53,103,129–133]. Self-assembly involves the design of a
scaffold-free method that mimics the behavior of embryonic stem cells and mini-tissues
can be defined as the smallest structural and functional component of a tissue [130].
Although this concept is still in its infancy, a number of in vitro and in vivo studies
have shown interesting perspectives that go beyond the principles of barrier membranes
and scaffolds. At the same time, there are numerous limitations to overcome, which
include production technology and bioinks optimization [134]. Photo-crosslinkable hy-
drogels, gelatin-methacryloyl hydrogel and poly (ethylene glycol) dimethacrylate have
been proposed as basic materials in bioprinting, and the optimization of printing capacity,
mechanical stability and cytocompatibility has been tried by testing different extrusion
parameters and crosslinking methods [134].
Wang et al. [135] proposed an active tissue engineering scaffold for the repair of
bone defects. This material was constructed with a 3D-BG scaffold composite, MSC and
nanoparticles loaded with the BMP-2 gene (BMP/CS). The scaffold was subsequently
implanted in the alveolar bone defect of rhesus monkeys and its capacity for osteogenesis
was assessed. The authors found that this scaffold model promoted bone healing, with
enhanced osteogenic properties in vivo [135].
Lin et al. [136] developed a biomimetic microfiber system, able to withstand functional
load, to help regenerate PDL. Straight, collagen-based waveform microfibers to guide
the growth of PDL cells were prepared by extrusion and a laminar flow-based bioreactor
was used to generate fluid shear stress. PDL cells were seeded on those microfibers [136].
The authors found that microfibers maintained the viability of PDL cells and showed an
improved tendency to promote healing and regeneration under shear stress.
Vurat et al. [137] developed a multicellular micro-tissue model similar to the periodon-
tal ligament-alveolar bone biointerface. The periodontal ligament layer was modeled using
methacryloyl gelatin bioink (Gel-MA) and ligament fibroblasts. The alveolar bone layer
was modeled using a composite bioink composed of Gel-MA and HA-magnetic iron oxide
nanoparticles (Gel-MA/HAp-MNPs), and osteoblasts [137]. The bioprinted self-supporting
microfabric was cultured under flow in a microfluidic platform for more than 10 days,
without significant loss of shape fidelity. Confocal microscopy analysis indicated that the
encapsulated cells were homogeneously distributed inside the matrix and maintained their
viability for more than 7 days under microfluidic conditions. Preliminary interaction study
with tetracycline indicated absorption of the drug by cells inside the 3D microfabric [137].
Li et al. [138] developed a system for vascularized bone regeneration; they combined
ethosomes loaded with deferoxamine (DFO) (Eth) with Gel-MA/gellan gum methacrylate
(GGMA) to fabricate 3D printed scaffolds by photo and ionic crosslinking. This system had
good cytocompatibility, generated sustained release of DFO, which significantly promoted
Membranes 2022, 12, 902 15 of 21

endothelial cell migration and tube formation, mineralized matrix deposition, and alkaline
phosphatase expression [138]. The scaffold was later placed in a rat cranial defect; the
signaling pathway of inducible hypoxia factor 1-α (HIF1-α) has been activated, indicating
that the composite scaffold could promote angiogenesis and bone regeneration [138].
Although 3D bioprinting has been identified as a technology capable of changing
the paradigms of regenerative and reconstructive periodontal therapy, it still finds itself
at the beginning of the road. Of course, further studies to translate in vitro models to
experimental animals and, subsequently, why not, to human subjects, are an absolute
necessity for the establishment of manufacturing and implementation protocols.

5. Future Directions
Periodontal regeneration involves a high degree of intricacy due to the complex nature
and architecture of the periodontal apparatus. The coordination of the components, gingiva,
periodontal ligaments, alveolar bone and root cementum, is of crucial importance in the
complete regeneration of periodontal structures. In addition, the periodontal system is
susceptible to microbial flora, as well as to local and systemic risk factors for periodontal
disease. Translating in vitro and in vivo models into clinical models on human subjects
is the main challenge associated with the development of technologies. Moreover, the
present studies pay little attention to antimicrobial and immunomodulatory strategies. The
integration of antimicrobial drugs and/or biomaterials with immunomodulatory capacity
is an extremely interesting future direction.
An interesting approach that is still in its early stages is the reconstruction of highly
organized living tissues and tissue interfaces in complementary converged 3D printing/bio-
printing technologies, integrated into a single biofabrication platform, called multi-technology
manufacturing. This field can generate scaffold systems with high print fidelity, spatial control
over cell distribution and improved biomechanical properties, without compromising cell
viability and functionality [139].
Studies already present in the literature have investigated the association of hydrogel
printing with MEW [140]. The authors combined extrusion-based bioengineering and
MEW in a single biofabrication platform, which allowed the fabrication of living constructs
with spatial distribution of mesenchymal stromal cells, with improved functionality and
biomechanics, without compromising cell viability or chondrogenic differentiation [140].
Diloksumpan et al. [141] also used convergent technologies to achieve hard-soft tissue
interfaces, using hydrogel and bioceramics.
Dos Santos et al. [142] combined co-axial electrospinning and 3D printing techniques to
generate zein-based bilayers as a potential platform for dual delivery of periodontal tissue
regeneration drugs. In vitro experiments showed that the two-layer constructs provided
sustained release of distinct drugs for 8 days and showed biocompatibility against human
oral keratinocytes (Nok-si) (cell viability > 80%), as well as antibacterial activity against
bacterial strains. distinct, including those from the red complex, such as Porphyromonas
gingivalis and Treponema denticola [142].
Liu et al. [143] associated directly deposition 3D printing of PCL/gel/nano-hydroxyapatite
(n-HA) scaffolds with polycaprolactone/gelatin nanofiber membranes (PCL/Gel) by electro-
spinning. The authors noted that after 20 weeks, the PCL/Gel/n-HA scaffold-treated sites
showed a higher degree of new bone formation than in the control group, indicating that this
is a favorable combination of GBR materials [143].
The fourth dimension was introduced in the bioprinting field; thus, smart scaffolds
have emerged, programmed to undergo shape or functional changes according to the
desired stimulation in time [144]. 3D-printed networks are stimulated by the environmental
factor to fold into tubes, mimicking vascular-like tissue constructs [31].
Other promising technologies include external magnetic field stimulation [145] or
atmospheric plasma functionalization [146]. Also, new perspectives are brought by real-
time monitoring of the printing process through combinations of artificial vision, inspection
Membranes 2022, 12, 902 16 of 21

sensors and feedback control systems supervised by high-efficiency artificial intelligence

algorithms and the biofabrication of truly functional tissue equivalents [16,139].

6. Conclusions
In response to the requirements of periodontal GTR and GBR, new feeding materials
and 3D printing methods have been developed, with the aim of accelerating production
and improving performance, with favorable in vitro and even in animal models results.
These aspects propel membrane printing and multiphasic scaffolds as a new and promising
approach in the reconstruction of periodontal tissues.
Of course, many limitations related to the bioavailability of bioinks, the mechanical
properties of the printed structure and its dimensional accuracy are still to be overcome.
Moreover, translating the obtained models to human subjects remains an important chal-
lenge. Improving digitally assisted techniques and biomaterials, together with the combi-
nation of CBCT investigations, can facilitate this translation, with the production of patient-
and site-specific scaffolds.
Tissue engineering is still in its early-stage development, due to the lack of in vivo and
clinical evaluation in periodontal defect models, the available data being limited.

Author Contributions: Conceptualization, I.-G.S. and S.M.S.; methodology, G.M., I.L. and D.-C.K.-N.;
resources, S.S. and M.-A.M.; writing—original draft preparation, I.-G.S., G.M. and S.S.; writing—
review and editing, M.-A.M., I.L. and S.M.S.; visualization, I.-G.S.; supervision, S.M.S.; project
administration, I.-G.S. and S.M.S.; funding acquisition, G.M. and S.S. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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