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Global Translational Medicine Gelatin-based cell carriers for tooth-germ organoids

the various stem cells used in tooth regeneration research, indicate the ability of GelMA MS as a scaffolding material
dental pulp stem cells (DPSCs) are considered an optimal for the formation of tooth organoids.
choice due to their self-renewal capabilities and potential While GelMA MS is promising for tooth organoid
2,3
for multilineage differentiation. DPSCs, derived from development, larger-scale studies, and pre-clinical animal
the pulp within the pulp chamber, play a crucial role in models are needed to validate their efficacy. Producing
replenishing odontoblasts, contributing to dentin repair, GelMA MS in high quantities requires a scalable and
and maintaining postnatal tooth homeostasis and repair. 2,4,5 reproducible method. The current production process is
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Recent advancements have highlighted tooth organoids labor-intensive, involving high-speed mixing, temperature
– three-dimensional constructs containing multiple cell control, nitrogen purging, heat crosslinking, and extensive
types, including DPSCs and dental epithelial cells – as washing steps, resulting in microspheres of variable sizes
a promising approach for tooth regeneration. Unlike and shapes that hinder reproducibility and scalability.
6-8
traditional two-dimensional cell cultures, organoids mimic Therefore, a simpler and more consistent production
the in vivo environment by providing a three-dimensional method is desired.
structure that supports complex cellular and structural Given the photo-crosslinkable nature of GelMA,
interactions essential for growth and differentiation. The encapsulating single or multiple cell types within a three-
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three-dimensional nature of organoids allows the presence dimensional HG is achievable. However, while encapsulated
of structural properties and chemical signals that promote cells in GelMA HG remain viable, limited nutrient and
accurate cell differentiation, making them a powerful waste diffusion can reduce cell viability and functionality
model for studying cellular behaviors outside of animal compared to cells cultured on HG surfaces. 17,18 To increase
models. Through cell-cell interactions, organoids support surface area, photo-crosslinked GelMA HG can be
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self-assembly, wherein cells drive scaffold aggregation to micronized into GelMA microparticles (GelMA MP),
form organoids, and self-organization, in which cellular which are easier to produce than GelMA MS. However, to
interactions organize different cell types to mimic natural evaluate the effectiveness of GelMA MP as a cell carrier, its
tissue structure, providing a functional representation of ability to support cell growth and differentiation must be
in vivo tissues (Figure 1E). 10-12 Fine-tuning the scaffolding directly compared to that of GelMA MS.
material is critical to successfully cultivating organoids that
accurately replicate the structure and function of natural Gelatin can also form microspheres (Gel MS), which
dental tissues. have been shown to support human mesenchymal stem
cell growth and functionality. 18,19 The production process
Hydrogels (HG), biomaterials that mimic the natural of Gel MS is simpler and more reproducible, allowing easy
cellular environment, are widely used in biomedical sorting by size. 18
applications due to their crosslinked hydrophilic polymer
networks, which allow them to retain large amounts In this study, we generated three types of gelatin-based
of water within their three-dimensional networks. 13-15 HG microparticles and compared hDPSCs cultured on
Gelatin, derived from collagen (the most abundant these carriers to cells encapsulated in GelMA HG. We
evaluated hDPSC growth and osteogenic differentiation
protein in the extracellular matrix), interacts with across the different carriers. Similar to GelMA MS, both
cells and forms HG upon crosslinking. Methacrylate GelMA MP and Gel MS supported the formation of
gelatin (GelMA), a derivative of gelatin modified with organoid-like structures. Among the four carriers, Gel
methacrylate anhydride, is photo-crosslinkable, allowing MS was most effective at supporting cell growth and
live cell encapsulation and crosslinking within GelMA differentiation, highlighting its potential as a superior
HG. Due to their biocompatibility and tunability, gelatin- scaffold for tooth organoid formation.
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based HG is frequently used in regenerative medicine,
including tooth regeneration. Studies have shown that 2. Materials and methods
GelMA microspheres (GelMA MS) can be loaded with
human DPSCs (hDPSCs) and injected into diseased dental 2.1. Synthesis of methacrylated gelatin
pulp to support regeneration. In addition, GelMA MS The synthesis and characterization of GelMA were
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have been shown to support the growth of hDPSCs and previously described. The one-pot GelMA synthesis
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human dental epithelial cells (hDECs) into organoid-like approach provides several notable benefits over
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structures. Co-culturing organoids formed from hDECs conventional methods, such as reduced reaction and
and hDPSCs resulted in fused organoids with cell-cell purification times, improved batch-to-batch uniformity,
interactions occurring between different cell types, closely and increased process control. Briefly, type A gelatin from
resembling the natural tissue environment. These results porcine skin (300 bloom, Sigma Aldrich, United States) was
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Volume 4 Issue 1 (2025) 68 doi: 10.36922/gtm.5897