The pulp-dentin complex is exposed to various stimuli, such as acids, sugars, and temperature shocks, which can affect the anatomy and function of the tooth [1]. Therefore, it is essential to construct a model of the pulp-dentin complex to study the relationship between external stimuli, dental materials, and the biological structure. There have been numerous tests, such as in vitro tests, animal models, and clinical examinations, that anticipate the biological and clinical viability of materials [2,3]. These tests offer invaluable insights into tissue responses towards materials [4]. In vitro culture models are popular because of their low cost and high efficiency, but they fall short in replicating the environment of native tissues [5,6]. Compared with 2D models, 3D models emerge as a more fitting option as they facilitate enhanced cell differentiation and tissue organization, which closely resemble the in-situ conditions.

Amidst the array of diverse 3D models [4,7,8], organs-on-chips (OoCs) have emerged as a promising avenue for investigating the intricate interfaces of organs while accessing real-time data. OoCs are bioengineered microdevices that unravel insights into human organ function. The scale of OoCs ranges from that of a USB drive to that of a standard 96-well plate, ingeniously mirroring the interconnectedness of multiple organs [9]. The engineering of OoCs finds its origins in the realms of microfluidics and tissue engineering, drawing synergistic principles from both disciplines [10]. OoCs utilize microdevices to realize well-defined microenvironmental parameters for various biological process studies. They also capitalize on the biomimetic paradigm, strategically integrating cell seeding within appropriate scaffolds with well-defined physical and biochemical cues. Living cells reside within these models, which mimic organ or tissue-level physiology [11], reproduce tissue structures and interfaces, facilitate liquid perfusion, and emulate physicochemical microenvironments. Moreover, the intrinsic benefits of microfluidic devices encompass remarkable precision, real-time imaging capabilities, and in vitro assessment of the biochemical and metabolic activities of living cells [6].

The development of organ-on-a-chip (OoC) technology has been ongoing for over a decade, and dentistry has been following this trend as well [6,12]. Researchers such as Niu et al. [13] have developed microfluidic chips that have microchannels and microchambers, which mimic the microstructure of dentinal tubules and culture odontoblasts, respectively. This model can be used to study the physiology and pathology of odontoblast processes. Sun et al. [14] designed a microfluidic device that consists of four different sizes of microtubes with varying lengths to investigate the invasion of Enterococcus faecalis. However, this design only mimicked a part of the tooth's anatomy, which is the structure of dentinal tubules. In 2020, França et al. [15] made a groundbreaking discovery in the dental field by developing the first tooth-on-a-chip. This device has two chambers separated by a dentin fragment and is used to study cell responses to dental materials. There are limitations to the design despite the groundbreaking effect. The fabrication of the microdevice relies on dentin fragments, which compromises consistency due to the different directions of the dentinal tubules. Additionally, the operation to fabricate the device is relatively complicated. While previous studies have focused on vasculogenesis and neurogenesis in the pulp-on-chip system [[16], [17], [18]], studies related to dentin are limited.

Dentin is the main hard tissue of the tooth, 70 % of components belong to inorganic substances, including dentinal tubules, odontoblast process, and intercellular substances. Mature dentin is a mineralized form of predentin matrix [19]. Formation of dentin mainly consists of odontoblastic differentiation, dentin matrix deposition, and dentin mineralization. We aimed to develop a novel simplified dentin-on-a-chip, which is more consistent and easier to operate to be a potential substitute for the dentinal fragment used in tooth-on-a-chip [15]. To overcome the limitations of previous studies, various aspects have been considered. (a) The material of choice for crafting OoCs is Polydimethylsiloxane (PDMS), which is an organic polymer derived from silicon and is predominantly employed to make microfluidics through the sophisticated technique of soft lithography [20]. (b) From a tissue engineering perspective, scaffolds play an important role in providing support for cells residing within OoCs. Gelatin methacrylate (GelMA) is a scaffold material that can encapsulate cells at 37℃, and maintain cell viability and proliferation [21,22]. GelMA's physical properties such as stiffness that can affect the differentiation of many stem cells, can be adjusted by changing GelMA and photoinitiator (PI) concentrations, making it a favorable material for tissue engineering applications. GelMA was used to create 3-dimensional (3D) biomimetic tooth bud models consisting of GelMA-encapsulated dental epithelial (DE) and GelMA-encapsulated dental mesenchymal (DM) cell bilayers, designed to facilitate DE-DM cell interactions, leading to ameloblast and odontoblast differentiation[23]. Khayat et al. [24]. used GelMA hydrogels to encapsulate hDPSCs/HUVECs for pulp tissue regeneration for clinically relevant applications. Thus GelMA was chosen to encapsulate stem cells in this study. (c) Stem cells from the apical papilla (SCAP) are recognized as a promising source of dental stem cells for tissue engineering due to their high potential of proliferation and mineralization[[25], [26], [27], [28]]. SCAP are precursor cells of dentin and dental pulp. Sonoyama et al. [29]. has concluded that SCAP are a unique population of postnatal stem cells distinct from DPSCs. Compared with DPSCs, SCAP demonstrated an elevated tissue regeneration capacity, higher telomerase activity, and an improved migration capacity. The osteo/odontogenic differentiation ability of SCAP was utilized to build the desired dentin-on-a-chip. Bioinks are substrates that may contain cells, matrix compounds, and signaling molecules within support materials such as hydrogels[30]. In our experiments, SCAP were mixed up within GelMA to form bioink and injected into the microfluidic chip.

So in this study, we have successfully designed a 3D microfluidic chip and established a 3D culture protocol for SCAP within the microdevice. Our approach involved using SCAP and GelMA to construct the 3D culture system, followed by inducing SCAP into osteo/odontogenic phenotype within GelMA to form dentin-like tissue. We also determined the appropriate cell seeding density at 2 × 104 cells/μL and GelMA concentration at 5 % to construct a simplified dentin-on-a-chip. Our model has the potential to be utilized as a biological model for screening dental materials.