Engineering innervated secretory epithelial organoids bymagnetic three-dimensional bioprinting for stimulating
epithelial growth in salivary glands
Christabella Adinea, Kiaw K. Nga, Sasitorn Rungarunlertb, Glauco R. Souzac,d, Joao N. Ferreiraa,e,f
Faculty of Dentistry, National University of Singapore, Singapore
Department of Preclinical and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, 73170, Thailand
cUniversity of Texas Health Sciences Center at Houston, Houston, TX, USAdNano3D Biosciences Inc., Houston, TX, USAe
Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
fNational Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USAabAbstract

Reference: Biomaterials 180 (2018) 52-66

Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully eﬀective due to the presenceof damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids
are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system,
the magnetic 3D bioprinting (M3DB), is tested to generate innervated secretory epithelial organoids from a neural crest
derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles(MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial diﬀerentiation stage wascompleted with ﬁbroblast growth factor 10 (4e400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis.
The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DBformed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-freespheroids. After diﬀerentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal,
myoepithelial, and neuronal. Fabricated organoids also produced salivary a-amylase upon FGF10 stimulation, and intracellular calcium mobilization and transepithelial resistance was elicited upon neuro stimulation with diﬀerent neurotransmitters. After transplantation, the SG-like organoids signiﬁcantly stimulated epithelial and neuronal growth in damaged SG.
It is the ﬁrst-time bio functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG
regeneration and the treatment of radiotherapy-induced xerostomia.

First report of a transplanted functional, innervated,salivary gland-like organoid produced through bioprinting from neural crest-derived, mesenchymal
stem cells.
Bioprinted organoids were transplanted into animals
where they significantly stimulated epithelial and neuronal growth in damaged salivary glands.
Demonstrate flexibility of applying magnetic 3D bioprinting for transplantation of SG-like organoids intothe excretory ductal area of developing SG glands
ex-vivo.
Organoids significantly rescued epithelial growth on
this acute SG fetal models of irradiation exposure.
SG-like organoid model can be further used as a toolfor cytotoxicity screening and mechanistic studies.
A significant step in using magnetic 3D bioprinted
cells to help advance tissue engineering towards
translational applications in regenerative medicine.
Bioprinting tool that can consistently control the sizeof the spheroids by tuning the concentration of MNP,
cell number, and the size of the magnet dots.

Results

Whole-mount immunoﬂuorescence staining of Salivary Gland (SG) spheroids

Quote“Nanoshuttle was instrumental for our 3D bioprinting techniques to eﬀectively produce spheroids on a reproducible mannerwith our adult stem cell cultures" said Joao N. Ferreira, DDM, PhD (Principal Investigator, BioprintME 3D Lab, ExcellenceCenter in Regenerative Dentistry, Chulalongkorn University, Bangkok, Thailandand Clinical Research Fellow, NIDCR,
National Institute of Health (NIH))
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