Boosting the angiogenesis potential of self-assembled mesenchymal stem cell spheroids by size mediated physiological hypoxia for vascularized pulp regeneration

Hypoxia is a pivotal factor in enhancing the vascularization potential of both two-dimensional (2D) cultured cells and three-dimensional (3D) cellular spheroids. Nevertheless, spheroids that closely mimic the in vivo microenvironment often experience excessive hypoxia, leading to the necrotic core and the release of toxic byproducts, ultimately impeding the regenerative process. To balance cell vitality and pro-angiogenic properties of cellular spheroids, this study investigates size-dependent hypoxia in stem cell spheroids utilizing an oxygen transfer finite element model. Subsequently, we develop 3D cultured stem cells from human exfoliated deciduous teeth (SHED) spheroids with regulated size-dependent hypoxia. Comprehensive assessments indicate that SHED spheroids, inoculated at a density of 50,000 cells, display moderate physiological hypoxia, which optimizes their pro-angiogenic potential, fusion capacity, and reattachment ability. Compared with SHED sheets, SHED spheroids enhance vascularized pulp regeneration more effectively with a tightly connected odontoblastic-like layer. Moreover, high-throughput transcriptome sequencing and RT-qPCR analysis further confirm the spheroids' ability to promote angiogenesis and odontogenic differentiation. This study not only introduces a practical and effective approach for regulating size-dependent hypoxia in cellular spheroids, and simultaneously enhancing cell vitality and angiogenic potential, but also paves the way for the clinical application of SHED spheroids in regenerative dental pulp therapies. STATEMENT OF SIGNIFICANCE: The core of three-dimensionally cultured cellular spheroids often experiences hypoxia, and maintaining a balance between the activity and functionality of long-term cultured spheroids in the inevitably hypoxic microenvironment remains a significant challenge. This study introduces a method to optimize the hypoxic conditions of SHED spheroids by employing a reaction-diffusion model, which modulates internal hypoxia to balance cellular viability and angiogenic potential. Compared to two-dimensional cell sheets, the optimized SHED spheroids with high cell vitality, angiogenesis potential, tissue integration and reattatchment ability show superior efficacy in promoting the formation of vascularized pulp-like tissue. This work offers valuable insights into the role of hypoxia in stem cell spheroids functionality and provides a foundation for further research into the optimization of stem cell-based therapies for multiple clinical applications.