Citation: Kilic Bektas, C.; Zhang, W.; Mao, Y.; Wu, X.; Kohn, J.; Yelick, P.C. Self-Assembled Hydrogel Microparticle-Based Tooth-Germ Organoids. Bioengineering 2022, 9, 215. https://doi.org/10.3390/ bioengineering9050215 Academic Editors: Teen-Hang Meen, Chun-Yen Chang, Charles Tijus and Po-Lei Lee Received: 8 April 2022 Accepted: 11 May 2022 Published: 17 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). bioengineering Article Self-Assembled Hydrogel Microparticle-Based Tooth-Germ Organoids Cemile Kilic Bektas 1,† , Weibo Zhang 2,† , Yong Mao 1 , Xiaohuan Wu 1 , Joachim Kohn 1 and Pamela C. Yelick 2, * 1 Department of Chemistry and Chemical Biology, Rutgers University, 123 Bevier Rd, Piscataway, NJ 08854, USA; cemile.bektas@rutgers.edu (C.K.B.); maoy@chem.rutgers.edu (Y.M.); xiaohuanwu2020@gmail.com (X.W.); kohn@dls.rutgers.edu (J.K.) 2 Division of Craniofacial and Molecular Genetics, Department of Orthodontics, Tufts University School of Dental Medicine, 1 Kneeland Avenue, Boston, MA 02111, USA; weibo.zhang@tufts.edu * Correspondence: pamela.yelick@tufts.edu † These authors contributed equally to this work. Abstract: Here, we describe the characterization of tooth-germ organoids, three-dimensional (3D) constructs cultured in vitro with the potential to develop into living teeth. To date, the methods used to successfully create tooth organoids capable of forming functional teeth have been quite limited. Recently, hydrogel microparticles (HMP) have demonstrated utility in tissue repair and regeneration based on their useful characteristics, including their scaffolding ability, effective cell and drug delivery, their ability to mimic the natural tissue extracellular matrix, and their injectability. These outstanding properties led us to investigate the utility of using HMPs (average diameter: 158 ± 32 μm) derived from methacrylated gelatin (GelMA) (degree of substitution: 100%) to create tooth organoids. The tooth organoids were created by seeding human dental pulp stem cells (hDPSCs) and porcine dental epithelial cells (pDE) onto the HMPs, which provided an extensive surface area for the cells to effectively attach and proliferate. Interestingly, the cell-seeded HMPs cultured on low-attachment tissue culture plates with gentle rocking self-assembled into organoids, within which the cells maintained their viability and morphology throughout the incubation period. The self-assembled organoids reached a volume of ~50 mm 3 within two weeks of the in vitro tissue culture. The co-cultured hDPSC-HMP and pDE-HMP structures effectively attached to each other without any externally applied forces. The presence of polarized, differentiated dental cells in these composite tooth-bud organoids demonstrated the potential of self-assembled dental cell HMPs to form tooth-bud organoid-like structures for potential applications in tooth regeneration strategies. Keywords: hydrogel microparticles (HMPs); tooth-bud organoids; GelMA microparticles; human dental pulp stem cells (hDPSCs); porcine dental endothelial cells (pDE); self-assembled organoid structures 1. Introduction Natural tooth development is the product of carefully orchestrated reciprocal interac- tions between the dental epithelium (DE) and the dental mesenchyme (DM) [1]. Facilitating proper DE–DM cell crosstalk is therefore essential to bioengineer constructs for functional whole-tooth regeneration. To date, tooth germ organoids have been created from easily ob- tainable mouse embryonic tooth-bud tissues and cells [2–4]. By contrast, human embryonic dental tissues are extremely difficult. if not impossible, to obtain. Using mouse embryonic DE–DM cells, efficient crosstalk was accomplished by combining DE and DM cell pellets directly within a collagen drop [5,6] that was subsequently cultured in semi-solid agar media [7], or by using a Trowell in vitro culture device [8]. These studies showed that the size of the regenerated teeth correlated with the size of the cell pellets within the collagen drop. Although embryonic mouse-tooth-derived regenerated teeth were comparable in size to natural mouse teeth, they were much smaller than human teeth. A similar approach Bioengineering 2022, 9, 215. https://doi.org/10.3390/bioengineering9050215 https://www.mdpi.com/journal/bioengineering