TYPE Review

PUBLISHED 22 February 2024

DOI 10.3389/fbioe.2024.1356580

The 3-dimensional printing for

OPEN ACCESS dental tissue regeneration: the
EDITED BY
Mona Kamal Marei,
Alexandria University, Egypt
state of the art and future
REVIEWED BY
Pravin D. Potdar,
challenges
Consultant, Mumbai, India
Jingang Xiao,
Southwest Medical University, China
Fengxiao Zhao 1,2,3, Zhijun Zhang 1,2,3 and Weihua Guo 1,2,3,4*
1
*CORRESPONDENCE State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University,
Weihua Guo, Chengdu, China, 2National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital
guoweihua943019@[Link] of Stomatology, Sichuan University, Chengdu, China, 3Department of Pediatric Dentistry, West China
School of Stomatology, Sichuan University, Chengdu, China, 4Yunnan Key Laboratory of Stomatology,
RECEIVED 15 December 2023 The Affiliated Hospital of Stomatology, School of Stomatology, Kunming Medical University,
ACCEPTED 06 February 2024 Kunming, China
PUBLISHED 22 February 2024

CITATION
Zhao F, Zhang Z and Guo W (2024), The 3-
dimensional printing for dental tissue
regeneration: the state of the art and Tooth loss or damage poses great threaten to oral and general health. While
future challenges. contemporary clinical treatments have enabled tooth restoration to a certain
Front. Bioeng. Biotechnol. 12:1356580.
extent, achieving functional tooth regeneration remains a challenging task due to
doi: 10.3389/fbioe.2024.1356580
the intricate and hierarchically organized architecture of teeth. The past few
COPYRIGHT
decades have seen a rapid development of three-dimensional (3D) printing
© 2024 Zhao, Zhang and Guo. This is an open-
access article distributed under the terms of the technology, which has provided new breakthroughs in the field of tissue
Creative Commons Attribution License (CC BY). engineering and regenerative dentistry. This review outlined the bioactive
The use, distribution or reproduction in other
materials and stem/progenitor cells used in dental regeneration, summarized
forums is permitted, provided the original
author(s) and the copyright owner(s) are recent advancements in the application of 3D printing technology for tooth and
credited and that the original publication in this tooth-supporting tissue regeneration, including dental pulp, dentin, periodontal
journal is cited, in accordance with accepted
ligament, alveolar bone and so on. It also discussed current obstacles and
academic practice. No use, distribution or
reproduction is permitted which does not potential future directions, aiming to inspire innovative ideas and encourage
comply with these terms. further development in regenerative medicine.

KEYWORDS

3D printing, regenerative dentistry, bioprinting, biomaterial, dentin-pulp complex,

periodontal regeneration, tissue engineering

1 Introduction
As an important craniofacial organ, teeth perform crucial functions including
mastication and pronunciation. Loss or damaged tooth can be caused by trauma,
bacterial infection, gene disease, or craniofacial cancer, posing great threaten to oral
health even general health. Though there are already available treatments to remove
damaged tissue and restore morphology and function of tooth to a certain extent, such
as resin restoration for caries, root canal therapy for infected pulp, dental implants for
edentulism, all of these clinical procedures have limited prognosis and fail to achieve full
replacement of native tissue. Therefore, regenerative therapeutic strategies are urgently
needed as alternative methods to create dental tissue substitutes with original structure and
function (Cooper, 2009; Sallum et al., 2019; Siddiqui et al., 2022).
The natural tooth features a sophisticated and hierarchically organized architecture.
Given that, the reproduction of complicated structure and integrated complex tissue remain
challenging (Duailibi et al., 2006; Yildirim et al., 2011). Nevertheless, the advancement of
biomaterials, further exploration of stem/progenitor cells, as well as innovative
biofabrication technologies are believed to facilitate dental tissue functional regeneration.

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FIGURE 1
Schematic diagram of the process for 3D printing dental tissue. (Created with [Link]).

TABLE 1 Techniques used in 3D printing of dental tissue.

Technique Advantages Disadvantages Application Reference

Extrusion-based • Extensive sources of • Limited printing resolution Dentin-pulp complex, alveolar bone, PDL, cementum, whole Gao et al. (2019)
printing material tooth, bio-root

• Low cost • Slow printing speed

• Shearing force unfavorable to

cells

Inkjet-based printing • Relatively fast printing • viscosity limitations of ink Dental pulp, alveolar bone Li et al. (2020a)
speed

• High resolution

• High cell viability

Laser-assisted printing • High printing speed • Expensive cost Dental pulp, bio-root, periodontal tissue, alveolar bone, PDL, Quan et al.
cementum (2020)
• High precision • Only light-curing materials

Currently, three-dimensional (3D) printing technology, or holds many advantages, such like high fidelity, customized
additive manufacturing, have developed tremendously, providing topographies, and superior production efficiency (Zhou et al.,
new breakthrough for tissue/organ regeneration (Mota et al., 2020). 2020; Vu et al., 2021). Up to now, numerous studies have
As a rapid prototyping technology, 3D printing precisely deposit focused on the application of 3D printing strategy in the field of
cells, materials, and bioactive agents into pre-defined locations in a regenerative dentistry (Ma et al., 2019).
“layer-by-layer” manner and construct intricated biomimetic The process workflow of 3D printing dental tissue is briefly
structures (Murphy and Atala, 2014). Compared with illustrated in Figure 1. The first step is to create a 3D model
conventional methods of tissue engineering, a 3D-printed scaffold containing the macroscopic and microscopic structure of the

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TABLE 2 Biomaterials used for dental tissue regeneration and their effects.

Type Material Bioink formulation Technique Effects Reference

Natural Collagen 0.2% collagen type I, 0.5% Inkjet Form vascular networks with hDPSCs and HUVECs ex Duarte Campos et al.
hydrogel agarose vivo (2020)

4% collagen Extrusion Form aligned PDL fibers in vivo Lee et al. (2021b)

Chitosan Chitosan, sodium alginate, Extrusion Antimicrobial effect on P. gingivalis; promote the Suo et al. (2023)
carbon nanotube proliferation of hPDLCs

Chitosan Extrusion Promote cell adhesion and viability of hDPSCs EzEldeen et al. (2021)

Alginate 4% alginate, 20% gelatin Extrusion Promote cell adhesion, proliferation and osteogenic/ Yu et al. (2019)
odontoblastic differentiation of hDPSCs

β-TCP β-TCP/PLGA at 75:25 ratio Extrusion Promote cell adhesion, proliferation and osteogenic Cao et al. (2020)
differentiation of hDPSCs

β-TCP, TPP, CMC Extrusion Promote osteoblastic differentiation of DPSCs Fahimipour et al.
(2018)

MTA 0.5g MTA, 6 g PCL Inkjet Promote cell adhesion and growth of DPSCs Wu et al. (2016)

Calcium silicate CS, PCL FDM Induce odontogenic differentiation of hDPSCs Huang et al. (2018)

Strontium-doped CS, PCL Extrusion Enhance bone regeneration in vivo Wang et al. (2021)

Hydroxyapatite Nano-HA, sodium alginate, Extrusion Promote cell survival, proliferation and osteogenic Tian et al. (2021)
gelatin differentiation of hPDLSCs

GelMA GelMA GelMA conjugated with BMP- Extrusion Promote odontogenic differentiation of hDPSCs Park et al. (2020)
peptide

GelMA, CS Extrusion Promote odontogenic differentiation of hDPSCs Choi et al. (2022)

15% GelMA, 10% CS Extrusion Promote odontogenic differentiation of hDPSCs Lin et al. (2021b)

10% GelMA, bioglass Extrusion Promote osteogenic/cementogenic differentiation of Mei et al. (2022)
nanoparticles hPDLCs

Synthetic PLA PLA FDM Promote odontogenic differentiation of DPSCs Feng et al. (2021)
polymer
PCL PCL, reduced graphene oxide Extrusion Promote cell adhesion of DPSCs Park et al. (2021)

wPU wPU, boric acid FDM Promote osteogenic differentiation of DPSCs Hung et al. (2016)

dECM dECM DFCs-derived dECM, GelMA DLP, DIW regenerate periodontium with hDFSCs in vivo Yang et al. (2023)

Bone-derived dECM, β-TCP Extrusion Promote the osteo/odontogenic differentiation of hDPSCs Kim et al. (2022)

TDM TDM, 30% PCL Extrusion Promote cell adhesion, proliferation and odontogenic Huang et al. (2023)
differentiation of DFSCs

scaffold. The data for the model can be acquired by computed Eventually, 3D printed constructs are cultured in vitro or
tomography (CT) scanning for post-processing or directly by implanted in vivo for biological regeneration.
computer design, also known as computer-aided-design (CAD). Even if 3D bioprinting still faces some hurdles, it has become an
Based on the target tissue, suitable biomaterials and cells are attractive and promising option to tooth and tooth-supporting
selected and prepared, as well as other bioactive additions. Then, tissue regeneration. In this review, we summarized the bioactive
the developed inks (only biomaterial) or bioinks (incorporating materials, cells, and recent progresses in the field of 3D printing for
with living cells) are loaded into the 3D printer. The common 3D dental tissue, and then, we highlighted the challenges that have
printing techniques include extrusion-based printing, as well as appeared in the tooth regeneration, and envision future directions
several variants such like fused deposition modeling (FDM)and for regenerative dentistry.
melt electrowritten (MEW), inkjet-based printing and laser-
assisted printing (e.g., stereo lithography appearance, SLA;
digital light processing, DLP, selective laser sintering, SLS) 2 Biomaterials in 3D printing for dental
(Mota et al., 2020). These techniques have variable features, tissue regeneration
which should be taken into account for manufacturing
(Table 1). For cell-free printing, cell seeding is commonly Several types of bioactive material have been used to
performed after scaffold fabrication has been completed. manufacture 3D-printed constructs for dental tissue regeneration,

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mainly including hydrogels, bioceramics, synthetic polymers and 2.1.4 Bioceramics

decellularized extracellular matrix (dECM). For mimicking the Bioceramic materials, including β-tricalcium phosphate (β-TCP),
natural tissue microenvironment, hybrid bioactive ink or bioink hydroxyapatite (HA), bioglass, calcium silicate (CS) and so on, have
are also developed (Table 2). been well investigated as scaffold materials in traditional tissue
engineering manufacturing for their excellent biocompatibility,
favorable bioactivity and easy tailored characters (Baino et al., 2015).
2.1 Natural hydrogel Owing to the intrinsic similar to the inorganic composition of bone and
tooth, bioceramics are often used for the regeneration of hard tissues.
2.1.1 Collagen But they also have disadvantages of low fracture resistance, low flexural
As the most abundant component in extracellular matrices resistance and wettability (Raveau and Jordana, 2020).
(ECM), collagen is widely used as the scaffold material in tissue Typically, bioceramics often added as fillers to the polymer
engineering (Wang Y. et al., 2023). Collagen contains arginine- matrix to obtain ideal physicochemical and mechanical properties.
glycine-aspartic acid (RGD)-motifs, which capable of mediate A study conducted by Ahlfeld et al. (2023) indicated that varieties of
cell adhesion (Wu et al., 2021). Collagen is not only the main mineral fillers, including CaCO3, SrCO3, strontium-modified
component of dental pulp and periodontium, but also the hydroxyapatite (SrHAp) or tricalcium phosphates, integrated into
biomineralization matrix of dentin and bone. Thus, collagen- a PLGA matrix can remarkably modify the degradation behavior of
based bioink have been used for the reconstruction of dental printed scaffold and an increase of the specific alkaline phosphatase
pulp, periodontal ligament, and alveolar bone (Duarte Campos activity were observed. Besides, mesoporous calcium silicate is also
et al., 2020; Lee U.-L. et al., 2021; Li et al., 2022). Despite good used in hard tissue regeneration as a component of biomaterial inks
biocompatibility, flexibility, and low immunogenicity, the poor because of its ability to sustained release Si ions and other bioactive
mechanical property limit collagen’s use as bioinks. agents (Huang et al., 2018; Yeh et al., 2022). A bioactive strontium-
Incorporation with other hydrogel or organic/inorganic doped calcium silicate (SrCS) scaffold released Sr and Si ions even
materials may be a feasible approach to improve mechanical after 6 months and enhanced secretion of osteogenic-related
stability and broaden the application boundaries of collagen- proteins (Wang et al., 2021).
based bioink (Lee J. M. et al., 2021). Commonly, hydrogels are mixed with bioceramics particles for
enhanced mechanical stability and adjustable biological properties.
2.1.2 Chitosan Tian et al. explored a novel bioink composed of sodium alginate
Derived from exoskeletons and shells of crustaceans, chitosan is (SA), gelatin (Gel), and nano-hydroxyapatite (na-HA). The SA/Gel/
abundant in nature and known as a widely used scaffold material in na-HA hydrogel exhibited shear-thinning behavior, optimized
tissue engineering. Chitosan possesses high biocompatibility, equilibrium swelling rate and compression modulus, as well as
hydrophilicity, and controllable biodegradability (Szulc and significantly improved the osteogenic differentiation of human
Lewandowska, 2022). Moreover, a broad spectrum of antibacterial periodontal ligament stem cells (hDPLSCs) (Tian et al., 2021).
properties makes it unique advantage for the dental application (Li Y.
et al., 2020). Numerous studies have proven that 3D-printed chitosan- 2.1.5 GelMA
based scaffold can be employed for dental tissue regeneration due to Gelatin methacryloyl (GelMA) is a gelatin derivative modified
its tunable physiochemical properties and favorable biological by methacrylamide and methacrylate groups. Resembling the native
response (Suo et al., 2023). EzEldeen et al. (2021) explored the ECM, GelMA contains many RGD-sequences for cell attachment as
effects of chitosan source (animal vs. fungal), co-polymerization well as matrix metalloproteinase (MMP)- degradable motifs for cell
with gelatine, and crosslinking agent (3-glycidyloxyproply remolding (Yue et al., 2015). As a photo-crosslinked hydrogel,
trimethoxysilane, GPTMS or genipin) on scaffold properties and GelMA undergoes photoinitiated radical polymerization without
biological response. However, the low mechanical property and chemical crosslinkers that may be detrimental to cells (Klotz et al.,
sterilization strict the application of chitosan-based hydrogel in the 2016). For its tunable mechanical characteristics and suitable
bioprinting field (Lazaridou et al., 2022). Therefore, many attempts biological properties, GelMA is a versatile matrix that can be
have been made to enhance the mechanical strength of chitosan by vastly used in biomedical application (Sakr et al., 2022).
functional groups modification and co-crosslinking with other Numerous studies have proven that GelMA-based scaffolds can
molecules (Liu et al., 2021; Salar Amoli et al., 2022). promote cell viability and osteo/odontogenetic differentiation of
dental stem cells, including DPSCs, PDLSCs and SCAPs (Ha et al.,
2.1.3 Alginate 2020; Buyuksungur et al., 2021; Zhu et al., 2023).
As a natural biopolymer, alginate attracted scientists’ focus for Not only served as cell delivery vehicles in bioprinting, GelMA
its water solubility and rapidly cross-linked by calcium ions at room also plays a key role in drug and growth factor delivery due to its
temperature. Besides, alginate possesses proper mechanical biodegradable properties. Park et al. (2020) developed bone
property, tunable viscosity and acceptable printability, which morphogenetic protein (BMP) peptide-tethering bioink by
make it suitable for bioprinting technique (Gao et al., 2021). conjugating peptides to GelMA. The results showed that BMP-
However, the pure alginate lacks RGD motifs for cell attachment, mimicking peptide remained in bioprinted construct for more than
and the enzyme that cleave alginate chain is absent in human body, 50% after 3 weeks and robustly increased the expression of
leading to the uncontrollable biodegradation in vivo. Given that, odontogenic-related genes of hDPSCs.
alginate is often used with other materials to form composites for A study conducted by Choi et al. (2022) used two different types
bioinks (Rastogi and Kandasubramanian, 2019; Yu et al., 2019). of MTA (ProRoot MTA and Endosem Zr) combined with GelMA

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and evaluated the biological effects on hDPSCs. Although 3D-

printed MTA/GelMA scaffolds showed more calcium deposition,
elevated expression of odontogenetic-related genes was not
statistically significant. Lin et al. fabricated the composite scaffold
with CS reinforced-GelMA bioink. The CS/GelMA bioink is capable
of inducing odontogenic differentiation of hDPSCs. Another study
showed that the addition of mesoporous bioactive glass
nanoparticles significantly enhanced shape fidelity, surface
roughness, and bioactivity (Mei et al., 2022).

2.1.6 Synthetic polymer

Synthetic polymers are another kind of vastly used materials in
tissue engineering. They have the advantages of adjustable physical
and chemical properties, which makes it suitable candidates for the
dental tissue regeneration.
Polylactic acid (PLA) and poly(L-lactic-co-glycolic) acid (PLGA)
FIGURE 2
are well-established polymers for their biosecurity approved by Food Primary of human dental pulp stem cells (hDPSCs).
and Drug Administration (FDA) and excellent manufacturing ability.
Moreover, PLGA possesses tunable degradation rate by different the
ratio of lactic acid to glycolic acid, which can be employed as carries of
biomedical substance (Danhier et al., 2012). However, both PLA and Treated dentin matrix (TDM), also termed as “demineralized
PLGA undergo hydrolytic degradation and generates acid dentin matrix” (DDM), is a kind of specific dECM that referred to
degradation products that may trigger local inflammation in vivo, dentin matrix after a certain degree of demineralization
thus confine their application (Ramot et al., 2016; Li et al., 2017). treatment. With exposed dentin tubules and collagen fibrils,
Polycaprolactone (PCL) is also conventional scaffold material TDM released abundant dentinogenetic-related biomolecules
routinely used in 3D-printing and has desired biocompatibility and including COL-1, DSPP, DMP-1, TGF-β1, decorin, biglycan,
biodegradability. Nevertheless, the lack of cell-anchoring site and and created an inductive microenvironment for dentin
hydrophobic surface lead to the low bioactivity of synthetic regeneration (Guo et al., 2009; Li et al., 2011). Athirasala et al.
polymers (Place et al., 2009). Various attempts have been made for (2018) mixed the insoluble components of the DDM with
the optimization of biological properties, including surface alginate in 1:1 ratio, and then added extracted soluble dentin
modification, biomaterial coating, introduction of functional molecules, dramatically promoting hydrogel-encapsuled SCAPs
groups and so on (Bow et al., 2021; Feng et al., 2021; Park et al., 2021). differentiate into dentin. Besides, TDM particles are also used to
Given that these traditional polymers often require organic form particle-based bioink and regenerate specific dental tissue
solvent for dissolving in 3D printing process and cause solvent or whole tooth (Huang et al., 2023).
residue in the final construct, in recent years, waterborne
biodegradable polyurethane (wPU) have shown great potential as
3D-printing materials (Hung et al., 2016). However, very few studies 3 The stem/progenitor cells used in the
are found about PU application in the dental tissue regeneration. A dentistry 3D printing
study published recently employed PU scaffolds coated with boric
acid. The wPU/boric acid scaffold can trigger the osteogenic 3.1 Dental pulp stem cells (DPSCs)
differentiation of DPSCs without significantly reducing cell
viability and proliferation (Çelebi-Saltik et al., 2023). Dental pulp stem cells (DPSCs) was firstly isolated and identified by
Shi and Gronthos in 2000 (Gronthos et al., 2000). As the mesenchymal
2.1.7 Decellularized extracellular matrix stem cell, DPSCs possess self-renewal potential and multi-directional
dECM refers to the natural tissue with the removal of differentiation capacity, being capable of differentiating into
immunogenic cellular components while three-dimensional odontoblasts, chondrocytes, adipocytes, and neural-like cells in vitro
network of ECM and biological molecules were remained, and regenerate dentin-pulp complex in vivo (Gronthos et al., 2002; Wei
including various proteins and polysaccharides. Compared with et al., 2007). The typical microscopic morphology of human DPSCs is
other hydrogels, dECM provides not only mechanical support shown in Figure 2. Compared with bone marrow-derived
and adhesion cites for cells, but also biological cues to impact mesenchymal stem cells (BMMSCs), DPSCs express more
cell behavior by cell-ECM interactions (Zhang et al., 2022). odontoblast-related genes, such as ALP, DSPP, and DMP-1
Several studies have proven that dECM derived from dental (Yamada et al., 2006). Due to their wide sources and minimally
tissue/cells hold the capacities of recruiting stem cells, promoting invasive process (Ferrúa et al., 2017), DPSCs have already became a
osteogenesis, and forming pulp-like tissue (Smith et al., 2012; Heng kind of ideal seeding cells for tooth tissue engineering (Anitua et al.,
et al., 2016; Nowwarote et al., 2021; Tan et al., 2021). Due to the 2018; Liu P. et al., 2022). DPSCs loaded 3D-printed PLA/HA scaffold
rising demand for biomimetic microenvironments of 3D printed exhibited higher degree of mineralization than the cell-free scaffold
constructs, dECM is gradually becoming a promising bioink (Chen et al., 2021). Some studies have also used gene-modified dental
material (Kim et al., 2022; Yang et al., 2023). pulp stem cells for 3D printing (Wang W. et al., 2023).

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3.2 Stem cells from human exfoliated (Guo et al., 2009; Guo et al., 2012; Yang H. et al., 2019; Meng et al.,
deciduous teeth (SHEDs) 2022). Though limited literatures reported the application of DFSCs
in dental tissue bioprinting, it is gradually attracting more and
In 2003, Miura firstly found that exfoliated human deciduous more attention.
tooth contains a population of multipotent stem cells and named it
stem cells from human exfoliated deciduous teeth (SHED) (Miura
et al., 2003). Similar to DPSC, SHEDs are capable of differentiating 3.6 Others
into odontoblast, adipocytes and neural-like cells. Owing to the
immaturity of SHEDs, several studies suggested that SHEDs are In addition to the cells mentioned above, there also various kinds
more proliferative than DPSCs and BMMSCs (Nakamura et al., of stem/progenitor cells used for tooth and periodontal regeneration,
2009; Wang et al., 2012). In addition to the high proliferation rate such like bone marrow derived mesenchymal stem cells (BMSCs)
and multilineage differentiation potential, the noninvasive (Golafshan et al., 2023; Miao et al., 2023), Hertwig’s epithelial root
harvesting procedure also make SHEDs a promising cell source sheath (HERS) cells (Tang et al., 2022), dental papilla cells (DPCs),
for the regeneration of tooth, cartilage and neural tissue (Yang X. gingival fibroblast cells (GFs) (Wang et al., 2021; Liu et al., 2023) and
et al., 2019; Pereira et al., 2019; Mahdavi-Jouibari et al., 2023). induced pluripotent stem cells (iPSCs) (Kim et al., 2023).

3.3 Periodontal ligament stem cells (PDLSCs) 4 3D printing for dental tissue
regeneration
Periodontal ligament stem cells (PDLSCs), firstly isolated from
periodontal ligament by Seo in 2004, are progenitor cells of The natural tooth is composed of both hard tissue (dental
cementoblasts, periodontal ligament fibroblasts and alveolar bone enamel, dentin, cementum and alveolar bone) and soft tissue
osteoblasts. Seo also found that PDLSCs hold the potential to (dental pulp, periodontal ligament, gingiva). Tables 3, 4
generate cementum/PDL-like structure in immunocompromised summarized recent advancements in dental soft and hard tissue
mice (Seo et al., 2004). As one of the well-established stem cells printing respectively. Notably, natural tissues and organs typically
in the field of regenerative dentistry, PDLSCs have been considered exhibit compartmentalized architecture with an intrinsic integration
as ideal seeding cells of 3D printed scaffold that are capable of of diverse components, and the tooth follows suit. Therefore, Table 5
regenerating periodontal tissue both in vivo and in vitro (Park et al., presents the applications of 3D printing/bioprinting in the
2012; Lee et al., 2014; Daghrery et al., 2023). regeneration of composite tissue, including the dentin-pulp
complex, periodontal complex, as well as whole tooth
and tooth root.
3.4 Stem cells from apical papilla (SCAPs)

The stem cells from apical papilla (SCAPs), located on the 4.1 Soft tissue regeneration
exterior of the immature permanent tooth root foramen area,
plays an important role in the root formation and development. 4.1.1 Dental pulp regeneration
In 2006, Sonoma collected human root apical papillae from young Surrounded by rigid dentin walls, dental pulp is a loose
adults (18–20 years old) extracted third molars and isolated stem/ connective tissue composed of cells (odontoblasts, fibroblasts,
progenitor cells that express the mesenchymal stem cells marker: DPSCs), collagen, nerves and blood vessels. Retaining vital pulp
STRO-1 (Sonoyama et al., 2006). It is demonstrated that SCAPs hold is of great importance for tooth nutrient, repairment, and sensory,
the capacity to regenerate dentin-like and pulp-like tissue in vivo especially for immature permanent teeth. When dental pulp
(Sonoyama et al., 2006; Huang et al., 2010; Hilkens et al., 2017). infected, root canal therapy (RCT) remains the first-option in
Besides, SCAPs can also differentiate into cementoblasts in vitro, clinical practice. However, tooth after RCT is faced with
which hold the promise for periodontal regeneration (Ebadi problems such as persistent inflammation, discoloration, and
et al., 2023). increased fragility (Ricketts, 2001). While pulp revascularisation
presents a promising clinical approach, it fails to reconstruct
functional pulp-like tissue and leads to unexpected calcification
3.5 Dental follicle stem cells (DFSCs) (Chen et al., 2012). Therefore, stem cell-based regenerative
endodontic therapy has attracted the worldwide attention.
The dental follicle is an ectomesenchyme-derived connective To achieve cellular pulp regeneration, it is essential to create a
tissue that surrounds the enamel organ and dental papilla, playing a microenvironment conducive to stem cell proliferation and
key role in periodontal tissue formation and tooth eruption (Zhang differentiation. Hydrogel proves to be a fitting candidate owing
et al., 2019). Dental follicle stem cells (DFSCs) are mesenchymal to its resemblance to the natural ECM. Various hydrogel have been
stem cells isolated from dental follicle tissue and are considered as evaluated for pulp bioprinting, encompassing fibrin, hyaluronic
precursor cells of cementoblasts, osteoblasts and periodontal acid, GelMA and so on (Han et al., 2019; Nejad et al., 2021;
ligament cells (Morsczeck et al., 2005). The present researches Cunha et al., 2023).
have confirmed that DFSCs hold the capacity of regenerating Pulp vascularization is one of the key objectives as well as
bone, dentin, periodontal tissue and tooth root-like tissue in vivo challenges in functional pulp regeneration. Several strategies have

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TABLE 3 Applications of dental soft tissue printing.

Tissue Bioink Cells/biological Technique Results Reference

supplement
Dental Collagen, agarose DPSCsa, HUVECsa Inkjet Successful vasculogenesis in root canal in vitro Duarte Campos et al.
pulp (2020)

Dental GelMA microspheres hDPSCs DLP Regenerate full-length dental pulp with blood Qian et al. (2023)
pulp vessels and nerve in minipigs model

PDL Collagen PDLSCs Extrusion Fabricate waveform microfibers under shear Lin et al. (2021a)
stress

PDL Collagen hPDLSCsa/FGF-2 Extrusion Produce connective tissue between titanium Lee et al. (2021b)
implant and bone

PDL (wax mold) PDLSCs Extrusion Form organized PDL cells Kim and Park (2020)

Gingiva Alginate, gelatin GFsa/i-PRF Extrusion Regenerate oral soft tissue and enhance Yi et al. (2022)
angiogenic activity in vivo

Gingiva Acellular dermal matrix, GFsa Extrusion Increase the amount of keratinized gingiva in Liu et al. (2023)
gelatin, sodium alginate vivo
a
Cells composing bioinks.

been utilized including the sacrificial material method and co- Lin H.-H. et al. (2021) fabricated a collagen-based waveform
culture with endothelial cells (Athirasala et al., 2017). Duarte microfibers and evaluated the microcrimped scaffold under shear
Campos et al. (2020) injected collagen based bioink into prepared load (6 dyne/cm2). Compared with straight microfibers, waveform
root canal via a handheld bioprinter. The results of microfibers exhibited enhanced cell spreading and adhesion, as well
immunofluorescence showed successful vascularization in the as upregulated expression of periostin (a key molecule for
root canal without the shrink of bioink. However, these strategies extracellular matrix assembly).
have yet to demonstrate their feasibility in vivo. Kim and Park (2020) created microgroove patterns on the
Remarkably, in a recently published work, Qian et al. scaffold surfaces with different slice intervals. After 7 days
successfully fabricated DPSC-loaded GelMA microspheres via a culture, the μG-25 (25.40 µm intervals) showed the ability to
DLP printer. These cell-loaded microspheres have improved organize aligned PDL cells while the μG-6 (6.35 µm intervals)
stemness and higher multi-directional differentiation potential, leaded random organization. Similarly, Pilipchuk et al. (2016)
including angiogenic, neurogenic, and odontogenic employed 3D-printed scaffold combined with micropatterned
differentiation. The regeneration of vascularized and neutralized film to achieve regeneration and integration of alveolar bone and
pulp-like tissue was demonstrated by subcutaneous transplantation PDL. Their conclusions support that optimized surface tomography
in nude mice and in situ experiment in swine (Qian et al., 2023). induce specific orientations of PDL fibers in a more
predictable manner.
4.1.2 Periodontal ligament regeneration
Periodontal ligament (PDL) is a connective tissue that is mainly 4.1.3 Gingival regeneration
composed of collagen type I fibers and proteoglycans. Owing to the Gingiva is another key component of oral soft tissue, which is
firm attachment between cementum and alveolar bone and specific essential for oral structural integrity and dental aesthetics. Although
oriented collagen fibers, it provides mechanical stability and autogenous gingival grafts have been widely used to reverse gingival
attenuate the masticatory stress to protect tooth. Furthermore, recession and augment periodontal soft tissue, the lack of donor
PDL contains an abundance of blood vessels and nerve endings tissue sources, the complexity of the surgical procedures, and the
that provide nourishment to the cementum and alveolar bone. postoperative pain and numbness still hamper a good prognosis
Utilizing mesenchymal stem cells present in the periodontium, (Yang M. et al., 2019). Therefore, the tissue engineering strategy
repairment and remodeling of periodontal tissue are possible seems to be a new option for regenerative oral soft tissue
(Hurng et al., 2011). Hence, the PDL regeneration is an essential augmentation. Tang along with his team developed a kind of
part of periodontal regeneration. bioink composed of alginate, gelatin and injectable platelet-rich
3D printing holds its intrinsic advantage to form highly- fibrin (i-PRF), which can release various repair-related growth
arranged collagen fibers because of the exist of micro-strands and factors. In vivo experiments, 3D-printed constructs with 50%
precise control of cell position in the 3D network. It has been proven i-PRF showed more collagen formation and host tissue
that compared with cell-seeding bulk collagen, the cell-laden infiltration compared to the group without. In their latest study,
collagen by bioprinting showed significantly more lang they bioprinted acellular dermal matrix (ADM)-based hydrogel
periodontal ligament-like soft tissue on the surface of titanium composite and performed in vivo implantation in beagles,
implants (Lee U.-L. et al., 2021). demonstrating that GF-loaded ADM scaffolds significantly
To physically control the orientations of fiber bundles, so called promote keratinised gingival regeneration (Yi et al., 2022; Liu
“fiber-guiding scaffold,” has emerged and attracted increasing focus. et al., 2023).

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TABLE 4 Applications of dental hard tissue printing.

Tissue Bioink Cells/biological Technique Results Reference

supplement
Enamel HAp nanorods; resin — Extrusion Multi-scale ordered tooth crown Zhao et al. (2022)

Enamel Carboxymethyl chitosan, HAT-7 cells Extrusion Promote ameloblast differentiation and matrix Mohabatpour et al.
alginate mineralization in vitro (2022)

Dentin Biodentine, PCL hDPSCs Extrusion Apatite formation; promote odontogenic Ho et al. (2018)
proliferation

Dentin Calcium silicate/calcium hDPSCs/quercetin Extrusion Promote odontogenic differentiation Yeh et al. (2022)
sulfate, PCL

Dentin Fibrinogen, gelatin, DDM DPSCsa Extrusion Tooth-shaped dental construct in vitro Han et al. (2021)
particle

Dentin PLA DPSCs/titania coating FDM Templated biomineralization and odontogenic Feng et al. (2021)
differentiation of DPSCs

Dentin Bone-derived dECM, DPSCsa Extrusion Promote the osteo/odontogenic differentiation Kim et al. (2022)
β-TCP

Cementum PCL PDLSCs, growth factors Extrusion Form cementum-like layer on the dentin surface Cho et al. (2016)

Alveolar GelMA HERSa, DPSsa Extrusion Regenerate new bone in vivo Tang et al. (2022)
bone

Alveolar GelMA, β-TCP BMSCs Extrusion Enhance osteogenesis Luo et al. (2022)
bone

Alveolar
bone
β-TCP/alginate OsteoInk ™ Alveolarbone MSCs Extrusion Print scaffolds to fit the respective clinical defects Anderson et al.
(2022)

Alveolar GelMA, PEGDA PDLSCs Inkjet Regenerate new bone in vivo Ma et al. (2017)
bone

Alveolar 6% Mg-CS — DLP Regenerate new bone in vivo Qin et al. (2022)
bone

Alveolar silk fibroin, collagen, HA rh-EPO Extrusion Regenerate new bone and collagen fibers in vivo Liu et al. (2022a)
bone

Alveolar CSi-Mg10 — Extrusion Regenerate new bone in vivo Shao et al. (2018)
bone

Alveolar Collagen, silk fibroin, nHA KSL-W Extrusion Regenerate new bone in vivo Li et al. (2022)
bone

Alveolar GelMA, PCL DPSCsa Extrusion Promote OPN and OCN expression and CaP Buyuksungur et al.
bone deposition (2021)
a
Cells composing bioinks.

4.2 Hard tissue regeneration multi-scale highly ordered structure and strong mechanical
strength. Mohabatpour et al. (2022) combined
4.2.1 Enamel regeneration carboxymethylcellulose (CMC) and alginate to create a new
Dental enamel is the hardest tissue in the human body, bioink that induced the differentiation of dental epithelial cells
providing external protection for the tooth against masticatory (HAT-7) to enamel-forming cells and promoted Ca and P
forces. As a kind of non-vascular, acellular, highly mineralized deposition as well as matrix mineralization in vitro.
hard tissue, enamel is composed of substituted hydroxyapatite
and organic macromolecules (Nanci, 2013). Since the enamel 4.2.2 Dentin regeneration
organ disappears at the end of the tooth germ development, Human dentin is composed of hydroxyapatite (70% by weight)
there are no enamel epithelial cells in a mature tooth, resulting and an organic matrix (35% by weight) of collagenous and
in the lack of enamel regeneration ability. Thus, development of noncollagenous proteins, such as dentin sialophosphoprotein
alternative approaches to repair and regenerate enamel defects is (DSPP), dentin matrix protein-1 (DMP-1), osteopontin, and
much needed but remains challenging due to the highly-ordered osteocalcin (Zhang et al., 2014). Dentin regeneration is one of
hydroxyapatite structure. Zhao et al. (2022) used supergravity the most explored parts in the field of dental tissue engineering.
preparation technology and hydrothermal treatment to disperse 3D printing is capable of forming customed macro structures and
the Hap nanorods in the hybrid resin-based composites (RBCs), intricated interconnections. Compared with conventional cast-
developing a new bioactive ink. The printed tooth crown possesses molded hydrogel, 3D-printed alginate/gelatin scaffold can better

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TABLE 5 Applications of dental composite tissue printing.

Tissue Bioink Cells/biological Technique Results Reference

supplement
Dentin-pulp PCL, fibrinogen, gelatin, hDPSCsa Extrusion Regenerate patient-specific dentin-pulp Han et al. (2019)
complex hyaluronic acid, glycerol complex in vitro

Dentin-pulp PCL hDPSCs/bioglass, hyaluronic Extrusion Regenerate dentin and dental pulp in vitro Nejad et al. (2021)
complex acid

Dentin-pulp GelMA, dentin matrix — DLP Regenerate organized odontoblast layer and Cunha et al. (2023)
complex molecules tertiary dentin

Periodontal PCL with MgP hMSCs MEW Regenerate PDL, bone and bone-ligament Golafshan et al.
complex interfaces in vivo (2023)

Periodontal GelMA, dECM hDFSCsa DLP, DIW Regenerate PDL, bone and bone-ligament Yang et al. (2023)
complex interfaces in vivo

Periodontal PCL — MEW Regenerate PDL and bone Yao et al. (2022)
complex

Periodontal GelMA, sodium alginate BMSCsa/BMP-2, PDGF Extrusion Regenerate bone and gingiva in vivo Miao et al. (2023)
complex

Periodontal PCL, HA DPSCs, PDLSCs, ABSCs/ Extrusion Regenerate cementum, PDL and bone Lee et al. (2014)
complex growth factors

Periodontal GelMA DFSCsa DLP Functional periodontal regeneration in vivo Ma et al. (2023)
complex

Periodontal PCL Decellularized PDL cell sheet MEW Regenerate cementum, PDL and bone Blaudez et al.
complex (2023)

Periodontal PCL F/CaP coating MEW Regenerate cementum, PDL and bone in Daghrery et al.
complex vivo (2021)

Periodontal PCL hPDLSCs/F/CaP coating MEW Regenerate PDL and bone in vivo Daghrery et al.
complex (2023)

Periodontal PCL PDLSCs, fibroblasts SLS Regenerate PDL and bone in vivo Pilipchuk et al.
complex (2016)

Tooth root PLA DPSCs/HA coating FDM Higher degree of mineralization in vivo Chen et al. (2021)

Whole tooth PCL SDF-1, BMP7 extrusion Regenerate tooth-like structures in vivo Kim et al. (2010)

Bio-root TDM, PCL DFSCs extrusion Regenerate anatomically shaped bio-root in Huang et al. (2023)
vivo

Bio-root HA DFSCs/nano-HA whiskers DLP Regenerate personalized bio-root with Chen et al. (2023)
coating PDL-like enthesis formation
a
Cells composing bioinks.

promote the cell adhesion, viability, and osteogenic/odontoblastic In addition to bioactive molecules, Yeh et al. (2022) fabricated a
differentiation of hDPSCs (Yu et al., 2019). Besides, 3D printed mesoporous calcium silicate/calcium sulfate (MSCS) scaffold and
Biodentine/PCL scaffold exhibited a good apatite-forming ability added quercetin, a novel bioactive molecule which can reduce
and induced hDPCs proliferation and differentiation (Ho inflammatory mediator and inhibit osteoclast activity, into the
et al., 2018). printed scaffold. The composite scaffold not only possessed better
Incorporation of biological cues also helps to induce the physicochemical and biological properties but also enhanced
odontogenic differentiation of stem/progenitor cells, and then odontogenic and immuno-suppressive properties. Feng et al.
promote the secreting and mineralization of dentin matrix. The (2021) coated a 5 nm thick titania layer via atomic layer
extracts of dentin matrix, both soluble and insoluble molecules, deposition on the 3D printed PLA scaffold, and higher DPSCs
significantly enhanced odontogenic differentiation of SCAPs plating efficiency and upregulated expression of osteocalcin
encapsulated in bioprinted hydrogels (Athirasala et al., 2018). compared with uncoated PLA scaffolds were observed.
Similarly, Han et al. (2021) mixed DDM particles with
fibrinogen-gelatin to produce a DDM particle-based bio-ink, 4.2.3 Alveolar bone regeneration
which enhanced the dentin-oriented differentiation of DPSCs. Alveolar bone loss is often caused by periodontitis or peri-
Besides, the study by Kim et al. indicated that supplemented with implant inflammation. Regeneration of alveolar bone is one of the
bone-derived dECM can accelerate the odontogenic differentiation goals of periodontal therapy, and it is also a prerequisite for implant
of hDPSCs (Kim et al., 2022). restoration. Compared with other craniomaxillofacial bone

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constructs, alveolar bone scaffold design and manufacture as a induced soft and hard tissues are likewise seen as one of the viable
tooth-supporting tissue remains a challenge due to the structural strategies. Nejad et al. (2021) constructed PCL/45S5 bioglass (BG)-
complexity and geometric adaptability of tooth-root surface. PCL/hyaluronic acid (HyA) scaffold and evaluated the physicochemical
Bioceramics play a crucial role in the bone tissue engineering. properties of the hybrid scaffold. The results showed that PCL/BG
Qin et al. explored the effect of different pore dimensions (Ø 480, scaffolds had superior mechanical strength and surface roughness, and
600, and 720 μm) on mechanical properties, bioactive ion release, the PCL/HyA scaffolds possessed increased hydrophilicity for cell
and bio-dissolution of bioceramic scaffolds. The 600 μm group adhesion. Recently, Cunha et al. (2023) utilized 3D-printed
exhibited maximal new bone formation in the rabbits’ microgels doped with dentin matrix molecules for direct dental pulp
mandibular bone defects model (Qin et al., 2022). In another capping. Although both the microgel group supplemented with DMM
™
study, Osteoink , composed of hydroxyapatite and α-TCP, was particles and the MTA group showed the formation of organized pulp
utilized to develop calcium phosphate (CaP)-based bioceramic tissue, dentin-like tissue and new blood vessels, the presence of DMM
scaffolds for alveolar bone reconstruction. The printed scaffolds helped to promote dentin bridge formation and exhibited more tertiary
had a high degree of accuracy to fit the respective clinical defects for dentin deposition and attenuate less pulp necrosis.
which they were designed (Anderson et al., 2022). A newly
developed bioceramic material, ~10% Mg-substituted wollastonite 4.3.2 Periodontal complex regeneration
(Ca90%Mg10%SiO3; CSi-Mg10) was also used to fabricated Periodontitis is one of the most prevalent oral diseases in the
customed scaffolds for reconstruction of alveolar bone defect, world, resulting in periodontal attachment loss, alveolar bone
exhibited desired osteoconduction, osteoinductivity and adequate resorption, even tooth loss (Kwon et al., 2021). The goal of
biodegradation (Shao et al., 2018). periodontal treatment is to control the infection and reconstruct
GelMA is another well-established material for alveolar bone the functional tooth-supporting tissue. Guided tissue regeneration
reconstruction by virtue of its analogue to extracellular matrix and (GTR) is currently gold standard of periodontal tissue reestablishment
adjustable physicochemical properties. 3D-printed PCL/GelMA hybrid by a barrier membrane to allow selective repopulation of PDL cells
scaffolds yielded cell viability, osteogenic differentiation of DPSCs and and bone, yet it still suffers from various limitations as well as clinical
mineralization in vitro (Buyuksungur et al., 2021). Tang et al. (2022) variability (Karring et al., 1993). The periodontal complex comprises
combined HERS cells and dental papilla cells (DPCs) to mimic the multiple tissues, including periodontal ligament, cementum and
micro-environment of cell-cell interaction. Plenty of mineralization alveolar bone, forming a “sandwich-like” structure. Herein,
texture were observed when transplanted into the rat’s alveolar sockets. multiphasic scaffolds to mimic the cementum-PDL-alveolar bone
GelMA/PEGDA hybrid hydrogel were fabricated by controlling the complex has increasingly attracted the attention of researchers
volume ratio of GelMA-to-PEGDA. The results indicated that the 4/ (Ivanovski et al., 2014).
1 GelMA/PEGDA composite hydrogel exhibited best performance in In previous studies, Lee CH and his colleagues developed various
osteogenic differentiation and formed new bone tissue in vivo (Ma et al., growth factor-releasing scaffolds by loaded PLGA microspheres for
2017). Luo et al. (2022) developed GelMA/β-TCP hybrid scaffolds periodontal tissue regeneration. They initially achieved integrated
which showed good biocompatibility and mechanical properties, and cementum-like tissue on the surface of dentin with high expression
stimulated osteogenesis of BMSCs. of cementum matrix protein 1 (CEMP1), an important marker of
Furthermore, the supplement of growth factors, peptides, and cementogenesis (Cho et al., 2016). Their another study used multiphase
other bioactive substances undoubtedly endows 3D printed scaffolds for periodontium complex regeneration via spatiotemporal
constructs with superior biological properties, such as delivery of multiple bioactive cues, involving recombinant human
antimicrobial peptide KSL-W (Li et al., 2022), recombinant amelogenin, CTGF and BMP-2. They also demonstrated the
human erythropoietin (rh-EPO) (Liu H. et al., 2022) and different microstrands spacing have an effect on the generation of
graphene oxide (Park et al., 2021). integrated multiple tissues (Lee et al., 2014). Consistent with Lee’s
studies, Daghrery et al. engineered PCL scaffold with F/Ca coating and
revealed that aligned fibers strongly support ligamentogenesis and
4.3 Multi-tissue regeneration scaffold architecture with 500 μm strands spacing supports
osteogenesis. The results of Masson’s trichrome staining indicated
4.3.1 Dentin-pulp complex regeneration that the tissue-specific scaffold induced the regeneration of both soft
The dentin and dental pulp derived from dental papilla, which is and hard tissue in vivo (Daghrery et al., 2021; Daghrery et al., 2023).
formed during tooth germ development and participate in tooth Furthermore, a novel composite hydrogel was designed as cell-laden
activities together as the dentin–pulp complex (Abbass et al., 2020). bio-ink, encapsulating BMP-2 and PDGF that effectively facilitate
Dentin offers durable shielding for the pulp, whereas the pulp alveolar bone regeneration and promote gingival healing respectively
supplies essential nutrients for the dentin and generates tertiary in the periodontal defect model of beagles (Miao et al., 2023). Ma et al.
dentin as a response to caries, mechanical injury, or acid erosion. (2023) reported biomimetic periodontium patches (BPPs) by DLP
Therefore, the regeneration of vital dentin-pulp complex is the ideal technology. The BPPs featuring correctly oriented fibers and the
target for endodontic medical treatment. assessment of clinically functional properties proved the regenerative
It has been widely studied that the fate of stem cells is closely periodontium can stand orthodontic migration force to achieve stable
associated with ECM characteristics (Liu et al., 2018). In this regard, tooth movement.
Han et al. (2019) developed hDPSCs loaded bioink with different Another important challenge to be solved is the interface
fibrinogen content to print dentin and pulp tissues (F20 for dentin, integration. Soft-to-hard tissue interfaces have always been a
F5 for dental pulp) respectively. Bilayer scaffolds with region-specific tricky problem in tissue engineering for the difficulty to

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reproduce interface enthesis (Calejo et al., 2019). To overcome the printed scaffold with PCL and TDM. Notably, the TDM scaffolds
lack of adhesion between newly formed PDL and bone, Blaudez et al. combined with DFSC sheets served as an analogue to natural tooth
(2023) designed a decellularized construct consisting of consisting of root and with satisfied angiogenic capacity in orthotopic
PDL cell sheet and PCL scaffold for tissue-specific periodontal transplantation in beagles (Huang et al., 2023).
regeneration. Before decellularization, the biphasic construct was Besides, a study by Chen et al. fabricated personalized bio-root by
cultured for 24 h to allow cell sheet adhesion and enhance the DLP printing of hydroxyapatite bioceramic. Bioceramic sintered at
cohesion between periodontium and bone part. Yao et al. (2022) 1,250°C exhibited almost twice elastic modulus than natural
introduced a transition region with 500-µm filament spacing, while decellularized dentine, which significantly enhanced the
the bone region was 250-μm filament spacing and the PDL region physicochemical properties of Hap. The nano-HAw (nano-
was consisted of aligned PCL filaments. The transition region hydroxyapatite whiskers) coating improved both mechanical property
separated the tissue-specific regeneration in the other two parts and surface hydrophilicity, and periodontal ligament-like enthesis
at the early time and promoted cellular cross-communication later formation in-situ transplantation in rat alveolar fossa (Chen et al., 2023).
between two regions. The insertion of ligamentous fibers towards
the bone and calcium gradient were observed in the transition space.
Similarly, Golafshan et al. (2023) fabricated a tri-layer scaffold in 5 Discussion
which PCL matrix with MgP was for bone, highly aligned PCL fibers
were presented for PDL and random PCL fibers were printed to Although restorative treatment is still the mainstream treatment,
mimic the bone-to-PDL interface. The tri-layer scaffold showed regenerative treatment is gradually occupying a place in dentistry
enhanced mechanical stability and achieved coordinated clinical practice. Notably, 3D printing technology is a promising
periodontal tissue regeneration in the rodent model. Moreover, a solution in the field of regenerative medicine. 3D printing holds
recent study reported a biomimetic periodontal module with high inherent advantages of construct geometric topologies that affect cell
architectural integrity. The module provided high-precision behavior and reproduce intricated microstructure to mimic the
topographical cues and biochemical environment conducive to natural tissue/organ physiochemical and biological properties.
regulating cell behavior and achieving periodontal regeneration In general, the materials used for 3D printing-based tooth
with the enthesis of the bone-ligament interface and well-aligned regeneration should conform the following characteristics: 1)
fibers in a beagle model (Yang et al., 2023). bioactivity with stem/progenitor cells; 2) ease of processing; 3)
proper mechanical properties that match the corresponding
4.3.3 Whole tooth/bio-root regeneration tissue; 4) ideal physical characteristics, such like porosity, surface
Whole tooth regeneration with natural tooth morphology and roughness, wettability, and viscosity. The types of biomaterials
function is the ultimate goal of dental regenerative medicine. Back in commonly used in 3D dental printing are summarized above.
2010, Kim et al. fabricated anatomically shaped human molar Currently, composite materials are gaining increasingly focus for
scaffolds and rat mandibular incisor scaffolds by 3D printing and the single material fail to yield heterogeneous constructs. Besides,
performed orthotopic implantation of rat incisor scaffolds, combined biological agents are integrated to the printed constructs for
with the delivery of SDF1 and BMP7. After 9 weeks, the histological multifunctional biological effects, such like growth factors,
results revealed regeneration of the PDL-like tissue and new bone at mRNA, exosomes, and drugs (Zhou et al., 2018).
the interface between the scaffolds and native alveolar bone (Kim Dentoalveolar tissue exhibits compartmentalized architectures with
et al., 2010). While it is possible to replicate the morphology of natural structural integrations. Though studies referred to in this review has
teeth, grafts often struggle to form a strong enough periodontal bond achieved regeneration of dental tissues to varying degrees, there are
with the host jawbone in a short period of time, which can challenges need to be addressed, such as adequate vascularization,
compromise their ability to withstand substantial occlusal forces. neuralization, and internal integration into the host tissue.
Tooth germ recombination appears to be another viable path to Moreover, for the acellular hard tissues subjected to strong
achieving whole tooth regeneration (Zhang and Yelick, 2021). Studies mechanical loads, the balance between prolonged mineralization and
have been conducted to generate bioengineered tooth germs by the scaffold degradation is still a tackle problem. The regeneration of a
reconstruction of dental epithelial and mesenchymal cells (Ono et al., single tissue cannot meet the needs of functional tooth reconstruction,
2017). However, it is still tough to regenerate eruption-competent herein, multi-tissue biofabrication of dental tissue has emerged as a
tooth germs hence the epithelial-mesenchymal transition during prominent area of investigation. The utilization of multiphasic scaffolds
tooth development is yet to be completely unveiled and replicating with designed tissue-specific organization for detin-pulp and
spatiotemporal interactions between different cells remains periodontal complex have been discussed above. However, scaffolds
challenging. 3D bioprinting technology is thought to offer new fabricated in this way usually lack interphasic cohesion and lead to
possibilities for bioengineered tooth germ due to its ability to compromised stability of printed constructs.
precisely control microstructures at the cellular level. To better mimic natural tooth tissues and achieve functional
Therefore, fabricating bioengineered roots, also known as “bio- tooth reconstruction, further research should be undertaken in
root,” and then restoring with artificial crown seem to be a much several areas. One is to develop novel 3D printing technique with
more feasible approach. Bio-root, referred to the bioengineered higher printing resolution. Up to now, extrusion-based 3D printing
tooth root, was firstly proposed by Sonoyama in 2006 and has is the most used printing technique in the field of tissue engineering
demonstrated the ability to regenerate dentinal tubule-like and by virtue of easy access to material and low cost, but the printing
periodontal ligament-like structures in swine model (Wei et al., precision is not comparable to laser-assisted printing. Secondly, the
2013). Up to recently, Huang et al. developed a personalized 3D- regulation of stem cell fate by the biomechanical properties of the

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extracellular matrix needs to be further explored, which will inspire administration. WG: Funding acquisition, Supervision,
the cell-matrix interaction in the printing architecture. The Writing–review and editing.
emerging 4D printing is capable of creating scaffolds that can
create controlled dynamic cellular microenvironment, providing a
potential tool for recapturing the natural process of tooth genesis Funding
and restoration (Wang et al., 2022). Another direction of
development lies in immunomodulation for 3D printed The authors declare financial support was received for the
constructs. The biomaterial scaffolds often trigger the host research, authorship, and/or publication of this article. This work
immune rejection in vivo, leading to the chronic inflammation was supported by the Nature Science Foundation of China
and eventually regeneration failure (Whitaker et al., 2021). Only (82270958) and the Major Science and Technology Projects in
a few studies have reported the immune effects of 3D printed dental Yunnan Province (202302AA310038).
substitutes, including modulation of inflammatory factors and
macrophage polarization.
Whilst the laboratory studies about 3D printing for dental tissue Conflict of interest
are encouraging, they are yet to be translated into clinical practice.
There have only been a limited number of studies which have The authors declare that the research was conducted in the
reported on the clinical evaluation of 3D-printed constructs in absence of any commercial or financial relationships that could be
the human body. To bridge the gap between laboratory and construed as a potential conflict of interest.
clinical applications, further safety and function tests are urgently
required. In conclusion, 3D printing technology displays promising
translational and therapeutic potential for the regeneration of dental Publisher’s note
tissue, which lays a strong foundation for regenerative medicine.
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
Author contributions organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
FZ: Writing–original draft, Conceptualization, Investigation, claim that may be made by its manufacturer, is not guaranteed or
Visualization. ZZ: Writing–review and editing, Project endorsed by the publisher.

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