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Tang, et al.
epithelial cells and mesenchymal cells derived from function changes in vivo [16,17] . GelMA containing the
embryos to rebuild bioengineered tooth germ . Although characteristics of natural and synthetic biological materials,
[5]
they generated a tooth with complete anatomical structure, can be cross-linked and solidified into gel by ultraviolet
[18]
the embryonic stem cells confined their widespread light under the action of photoinitiator . Besides, GelMA
applications in clinical practice. Besides, the bioengineered has excellent biocompatibility, biodegradability, cell
tooth was irregular in both morphology and quantitative response characteristics, and the 3D structure suitable for
terms, and epithelial-mesenchymal interaction (EMI) cell growth and differentiation . In recent years, GelMA
[19]
was also in no small measure overlooked as the in vitro has been widely used in 3D cell culture, tissue engineering
experiment was conducted with two-dimensional cell and 3D bioprinting [20-23] . Stem cells and extracellular matrix
culture. To optimize the EMI process, other researchers tried were encapsulated in GelMA and poly(ethylene glycol)
building a biomimetic 3D tooth bud model by combining dimethacrylate composite hydrogel for alveolar bone
[24]
cell patch with dental epithelial and mesenchymal cells repair . 3D-printed cell/GelMA constructs could mimic
loaded with gelatin methacrylate (GelMA) hydrogel [6,7] . the micro-environment in vivo. Meanwhile, it provides
With respect to the foregoing, it could be fraught with excellent conditions for cellular activity and optimized
[25]
difficulties to realize the regeneration of tooth with both cell-cell interactions . Therefore, according to the spatial
crown and roots for now. Tooth root regeneration seems relationship between HERS cells and DPCs during tooth
more feasible than whole tooth regeneration in clinical germ development, we designed structural model of these
view. During tooth development, when the formation two kinds of cells and prepared two bio-inks using GelMA
of crown is nearly complete, the root begins to develop. hydrogel for 3D bioprinting (Figure 1). An alveolar bone
The inner and outer enamel epithelia proliferate around defect model was applied to test the osteogenesis effects
cervical loop and form a bilayered epithelial sheath known of 3D printed co-culture constructs. This study puts
as Hertwig’s epithelial root sheath (HERS). Considering forward a new method to recombine HERS cells and
that the inner cells of HERS initiate the differentiation of DPCs through 3D bioprinting, which could contribute to
odontoblast to form root dentin, HERS is indispensable to the investigation of EMI research in the future.
root development. For instance, if the continuity of HERS 2. Materials and methods
were damaged, it could not induce dental papilla cells
(DPCs) to differentiate into odontoblasts, which could 2.1. Ethics statement
result in dentin defects. In addition, if the epithelial root
sheath failed to break at specific time and adhered to the Animals were purchased from Dashuo Experimental
surface of the root dentin, the dental follicle cells could not Animal Co. Ltd. (Chengdu, China). All animal
differentiate into cementoblasts to form cementum [8,9] . In a experiments in this study were approved by the
previous study, we found that the HERS spheroids could Research Ethics Committee of West China Hospital
promote HERS cell proliferation and optimize its biological of Stomatology, Sichuan University (permit no.
function . The experiment verified the capability of WCHSIRB-D-2021-551).
[10]
HERS spheroids in the induction of DPCs differentiation 2.2. Cell isolation and culture
by mixing HERS spheroids with DPCs, but the interaction
between cells was also limited to some extent. The issue Primary HERS cells and DPCs were isolated from the
that needs to be addressed is to maximally maintain the first molar germ in the maxilla and mandible of 8-day-
epithelial characteristics of HERS and thereby inducing postnatal Sprague-Dawley (SD) rats (Figure S1), and
DPCs to form new mineralized tissue. the specific method was mentioned in the previous
Three-dimensional (3D) bioprinting is the studies [26,27] . After being sheared to fragments, the tissue
technology using a bioprinter to produce the pre-designed was digested by type I collagenase and dispase (Sigma-
3D structure made of different kinds of biomaterials and Aldrich, USA) solution at 37℃ for 10–20 min.
transit them to biologically active tissues or organs . This HERS cells were cultured in epithelial cell medium
[11]
cutting-edge technology enables the realization of new (ScienCell, USA) containing 1% epithelial cell growth
ideas for tissue engineering and shows great potential in supplement, 2% fetal bovine serum, and 1% penicillin/
biomedical applications due to its high precision and high streptomycin solution. Mixed with mesenchymal cells,
throughout [12,13] . Fabricated 3D fibrous scaffolds exhibited HERS cells in passage 0 were treated with 0.25% trypsin-
excellent ability to guide cell to arrange along the fiber EDTA (Gibco, USA) for 2–3 minutes, then purified
axis . In addition, “time” was incorporated into 3D HERS cells were obtained after 2–3 times of differential
[14]
bioprinting as the fourth dimension . In 4D bioprinting, digestion. The adherent HERS cells were processed into
[15]
the functionality of printed construct would change along cell suspensions under the treatment of TrypLE Express
with time under external stimulus, which might serve as a Enzyme (Thermo Scientific, USA) for 10–15 min.
useful tool for bio-fabrication in stimulating the biological DPCs were cultured in alpha-minimum essential medium

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