Dental pulp derived stromal cells or dental pulp stem cells (DPSCs) are of ectomesenchyme origin and exhibit enormous in vitro trans-differentiation capability depending on the culture conditions (Kawashima, 2012). Isolation protocols for the successful establishment of primary cultures of DPSCs include both enzymatic procedures and explant methods (Ferrúa et al., 2017, Pilbauerova et al., 2019). Conventionally, the isolation and expansion of DPSCs are achieved in surface-treated plastic flasks in 2-dimensional monolayer culture (2D culture) methods. Since 2D monolayer culture platforms are simple, cost-effective, convenient, and allow easy cultivation of cells (Ryu et al., 2019, Xu et al., 2020), most of the DPSCs based pre-clinical studies for cell therapy employ two- dimensional (2D) monolayer cultured stem cells model.

The choice of an appropriate culture method or model to simulate the in vivo microenvironment remains an important factor to maximize the therapeutic efficacy. 2D monolayer culture methods can provide adequate cells for clinical therapies, however, cells in physiological conditions do not grow in monolayers. The cells in the conventional 2D cultures lack a well-organized tissue architecture and important cell-cell, cell–environment interactions indispensable for the maintenance of stem cell characteristics including stemness properties, proliferation, and survival (Mirbagheri et al., 2019). Consequently, the therapeutic efficacy of the cells is impeded as they are deprived of important physical and chemical cues that regulate the cell fate (Mirbagheri et al., 2019, Yoon et al., 2018). Moreover, the dissociated monolayer cells from 2D culture exhibits lower biological efficacy, poor cell survival rate, lower engraftment rate and homing abilities which eventually hinder their clinical translation (Kim et al., 2023, Lee et al., 2021).

Over the course of time, various advances have been made in the field of tissue and cell culture techniques. One of the breakthrough innovations is the advent of 3D culture methods aiding in the generation of more accurate data and valuable insights in the field of stem cell regeneration. Three-dimensional in vitro culture techniques enable the formation of multicellular spheroids that are regarded as a closer cellular model of actual physiological parameters present in vivo. Cells grown in spheres are in close association with each other and their interaction with the extracellular matrix is also enhanced. These cellular interactions within the spheroids provide an environment or signal cues to each other much easier than in monolayer cultures where cell-to-cell contact and communication is limited. Therefore, the purpose of this study is to assess the biological and regenerative properties of the DPSCs 3D spheroids.

It is important that the spheroids simulate the in vivo microenvironment in terms of cell-cell interactions, matrix deposition, gene expression profile and nutrient exchange (Wang et al., 2020). In recent years, massive advancements have been made in developing strategies for three-dimensional cultures (Alhaque et al., 2018). Several scaffolds or scaffold-free 3D culture systems have been established for the generation of multicellular spheroids. Each system has its advantages and disadvantages and depending upon the experimental need any of the methods can be utilized. Scaffold-based methods rely on the utilization of synthetic or natural polymer-based hydrogel which provides an ideal environment for efficient spheroid formation. The methodologies for scaffold-based spheroid formation include micropatterned plates, matrix encapsulation, matrix on top, matrix embedded, microcarriers beads, and microfluidic devices (Ryu et al., 2019). However, scaffold-based techniques require prior preparation, matrix handling, expensive apparatus, equipment, and reagents (Antoni et al., 2015). The effective collection and spheroid separation from the scaffold remain laborious and challenging (Pinto et al., 2020). In this regard, we aimed to employ a simple scaffold-free approach that could facilitate the rapid multicellular spheroid formation of DPSCs via self-aggregation or self-assembly as well as convenient spheroids collection.

We evaluated the multicellular 3D spheroids of DPSCs for their structural integrity, morphology, viability, proliferation, stem cell phenotype, differentiation as well as regenerative abilities. Previously, our lab has demonstrated the higher propensity of dental pulp stem cells toward osteogenic lineage (Kumar et al., 2018, Raik et al., 2022). Here, we utilized animal model of bone defect to assess the in vivo regenerative ability of DPSCs from 2D and 3D culture. The multicellular spheroids generated under scaffold-free ultra-low attachment conditions provide a better platform for physiological studies and may show better clinical translation.