Regenerative Potential of Exosomes from 3D Spheroid-Cultured Stem Cells Compared with 2D Culture in Dentin–Pulp Regeneration: A Systematic Review

Purpose: This systematic review aimed to evaluate the current preclinical evidence on exosomes derived from three-dimensional stem cell culture systems and their direct or indirect relevance to dentin–pulp regeneration. Exosomes derived from stem cells have emerged as promising mediators of tissue regeneration because of their ability to transfer bioactive molecules that regulate cellular communication and tissue repair. Recent studies suggest that exosomes obtained from three-dimensional (3D) spheroid cultures may exhibit enhanced regenerative properties compared with those derived from conventional two-dimensional cultures. Methods: A systematic review was conducted following the PRISMA 2020 guidelines. Electronic searches were performed in PubMed, Scopus, and ProQuest databases to identify relevant studies investigating exosomes derived from 3D cell culture systems with potential relevance to dentin–pulp regeneration. Studies were screened according to predefined inclusion and exclusion criteria. Data extraction focused on cell sources, exosome isolation methods, characterization techniques, and biological effects related to dental pulp regeneration. Results: Ten studies met the inclusion criteria. The included studies demonstrated that exosomes derived from 3D spheroid cultures may enhance angiogenesis, support odontogenic differentiation, reduce inflammatory responses, and promote regenerative tissue repair. Several studies also reported increased expression of regenerative markers and improved cellular proliferation when compared with exosomes derived from conventional two-dimensional cultures. However, only a limited number of studies directly investigated dentin–pulp-related outcomes, while several provided indirect mechanistic evidence from non-dental preclinical models. Conclusion: Exosomes derived from three-dimensional culture systems show promising potential as a cell-free therapeutic strategy for dentin–pulp regeneration. These vesicles may improve tissue repair through enhanced biological activity. However, the current evidence remains mainly preclinical, heterogeneous, and partly indirect. Further standardized experimental studies and clinical investigations are required to validate their translational application in regenerative endodontic therapy. Plain Language Summary: Damage to dental pulp is commonly treated with root canal therapy, which removes infected tissue but does not restore the natural living function of the tooth. Regenerative dentistry aims to help repair the soft tissue and hard tissue inside the tooth. Exosomes are tiny particles released by cells. They carry helpful signals that can guide nearby cells to repair tissue, reduce inflammation, and support healing. Stem cell-derived exosomes have been studied as a possible treatment for dental pulp repair. Recent studies suggest that cells grown in rounded three-dimensional groups may release exosomes with stronger repair activity than cells grown in flat two-dimensional layers. This systematic review examined current studies on exosomes from three-dimensional stem cell cultures and their possible role in dentin–pulp regeneration. The findings suggest that these exosomes may help form new blood vessels, reduce inflammation, and support tissue repair. Some studies also showed effects related to dentin-forming cells. However, most available studies were performed in laboratories or animal models, and some were only indirectly related to dental pulp regeneration. More standardized studies and clinical research are needed before this approach can be used routinely in dental treatment. The infographic compares 2D and 3D spheroid cultures. 2D culture features a flat monolayer with limited cell interaction, while 3D culture enhances cell interactions, mimics ECM and supports physiological gradients and paracrine signaling. Exosomes from these cultures contain microRNAs, proteins and growth factors, with 3D conditions affecting their cargo and activity. Preclinical studies show biological effects like angiogenesis, odontogenic differentiation, anti-inflammatory effects and tissue repair. Angiogenesis supports endothelial activity and vessel formation. Odontogenic differentiation aids in forming odontoblast-like cells and dentin. Anti-inflammatory effects modulate immune responses and reduce pro-inflammatory cytokines. Tissue repair boosts cell survival, proliferation and ECM remodeling. Dentin-pulp regeneration offers potential for revascularization, dentin bridge formation, pulp vitality and cell-free therapeutic strategies.An infographic illustrating 2D vs 3D culture, exosome cargo, biological effects and dentin-pulp regeneration. Keywords: exosomes, three-dimensional culture, dental pulp stem cells, dentin–pulp regeneration, regenerative endodontics, extracellular vesicles