Pulpitis is a common and frequent disease in dental clinics. Although vital pulp therapy and root canal treatment can stop the progression of inflammation, they do not allow for genuine structural regeneration and functional reconstruction of the pulp–dentin complex. In recent years, with the development of tissue engineering and regenerative medicine, research on stem cell-based regenerative endodontic therapy (RET) has achieved satisfactory preliminary results, significantly enhancing its clinical translational prospects. As one of the crucial paracrine effectors, the roles and functions of exosomes in pulp–dentin complex regeneration have gained considerable attention. Due to their advantages of cost-effectiveness, extensive sources, favorable biocompatibility, and high safety, exosomes are considered promising therapeutic tools to promote dental pulp regeneration. Accordingly, in this article, we first focus on the biological properties of exosomes, including their biogenesis, uptake, isolation, and characterization. Then, from the perspectives of cell proliferation, migration, odontogenesis, angiogenesis, and neurogenesis, we aim to reveal the roles and mechanisms of exosomes involved in regenerative endodontics. Lastly, immense efforts are made to illustrate the clinical strategies and influencing factors of exosomes applied in dental pulp regeneration, such as types of parental cells, culture conditions of parent cells, exosome concentrations, and scaffold materials, in an attempt to lay a solid foundation for exploring and facilitating the therapeutic strategy of exosome-based regenerative endodontic procedures.

Introduction: Exosomes derived from M2 macrophages (M2-Exos) exhibit tremendous potential for inducing tissue repair and regeneration. Herein, this study was designed to elucidate the biological roles of M2-Exos in regenerative endodontic therapy (RET) compared with exosomes from M1 macrophages (M1-Exos). Methods: The internalization of M1-Exos and M2-Exos by dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) was detected by uptake assay. The effects of M1-Exos and M2-Exos on DPSC and HUVEC behaviors, including migration, proliferation, odonto/osteogenesis, neurogenesis, and angiogenesis were determined in vitro. Then, Matrigel plugs incorporating M2-Exos were transplanted subcutaneously into nude mice. Immunostaining for vascular endothelial growth factor (VEGF) and CD31 was performed to validate capillary-like networks. Results: M1-Exos and M2-Exos were effectively absorbed by DPSCs and HUVECs. Compared with M1-Exos, M2-Exos considerably facilitated the proliferation and migration of DPSCs and HUVECs. Furthermore, M2-Exos robustly promoted ALP activity, mineral nodule deposition, and the odonto/osteogenic marker expression of DPSCs, indicating the powerful odonto/osteogenic potential of M2-Exos. In sharp contrast with M1-Exos, which inhibited the neurogenic capacity of DPSCs, M2-Exos contributed to a significantly augmented expression of neurogenic genes and the stronger immunostaining of Nestin. Consistent with remarkably enhanced angiogenic markers and tubular structure formation in DPSCs and HUVECs in vitro, the employment of M2-Exos gave rise to more abundant vascular networks, dramatically higher VEGF expression, and widely spread CD31⁺ tubular lumens in vivo, supporting the enormous pro-angiogenic capability of M2-Exos. Conclusions: The multifaceted roles of M2-Exos in ameliorating DPSC and HUVEC functions potentially contribute to complete functional pulp–dentin complex regeneration.

Maintaining immune balance plays crucial roles in the development and prognosis of injured oral tissues. Serving as critical components of immune system, macrophages are essential in accelerating oral tissue repair and regeneration. This multipart review intends to present a comprehensive overview of recent advances and future perspectives of macrophage plasticity and polarization in oral tissue repair and regeneration, thereby enhancing the understanding of macrophage-induced immunotherapeutic effects and underlying mechanisms in oral diseases. The primary electronic databases consulted for this review included PubMed, Web of Science, and Scopus. The literature search strategy employed a comprehensive combination of keywords such as "macrophage polarization", "M1", "M2", "oral tissue repair", "periodontitis", "pulpitis", "osseointegration", "jawbone defect", and "immunotherapy". Both original research articles and authoritative review papers were selected. Furthermore, the reference lists of retrieved articles were manually examined to identify additional pertinent publications. Macrophages are derived initially from hematopoietic stem cells in the bone marrow and embryonic yolk sac progenitor cells. They encompass several polarization phenotypes, including M1, M2a, M2b, M2c, and M2d, and exert multifaceted functions by secreting soluble factors and insoluble exosomes. Encouragingly, robust immunotherapeutic potential and distinctive regulatory mechanisms of macrophages have been revealed for a variety of oral diseases, providing a breadth of perspectives and insights into the repair and regeneration of oral tissues. Orderly and sequential polarization of macrophages has been proposed as novel immunotherapeutic strategies and promising directions for treating oral diseases. An in-depth exploration of hidden mechanisms may contribute to structural regeneration and functional reconstruction of oral tissues.

Establishing an optimized regenerative microenvironment for pulp-dentin complex engineering has become increasingly critical. Recently, exosomes have emerged as favorable biomimetic nanotherapeutic tools to simulate the developmental microenvironment and facilitate tissue regeneration. This study aimed to elucidate the multifaceted roles of exosomes from human dental pulp stem cells (DPSCs) that initiated odontogenic differentiation while sustaining mesenchymal stem cell (MSC) characteristics in odontogenesis, angiogenesis, and neurogenesis during pulp-dentin complex regeneration. Differential centrifugation was performed to isolate exosomes from normal DPSCs (DPSC-Exos) and DPSCs that initially triggered odontogenic differentiation (DPSC-Od-Exos). The impact of these exosomes on the biological behavior of DPSCs and human umbilical vein endothelial cells (HUVECs) was examined in vitro through CCK-8 assay and Transwell migration assay, as well as assays dedicated to assessing odontogenic, angiogenic, and neurogenic capabilities. In vivo, Matrigel plugs and human tooth root fragments incorporating either DPSC-Exos or DPSC-Od-Exos were subcutaneously transplanted into mouse models. Subsequent histological, immunohistochemical, and immunofluorescent analyses were conducted to determine the regenerative outcomes. DPSC-Exos and DPSC-Od-Exos revealed no remarkable difference in their characteristics. In vitro analyses indicated that DPSC-Od-Exos significantly facilitated the proliferation, migration, and multilineage differentiation of DPSCs compared with DPSC-Exos. Furthermore, DPSC-Od-Exos elicited a more pronounced effect on the tubular structure formation of HUVECs. Consistently, Matrigel plug assays confirmed that DPSC-Od-Exos exhibited superior performance in promoting endothelial differentiation of DPSCs and stimulating angiogenesis in HUVECs. Notably, DPSC-Od-Exos contributed to complete pulp-dentin complex regeneration in human tooth root fragments, characterized by enriched neurovascular structures and a continuous layer of odontoblast-like cells, which extended cytoplasmic projections into the newly formed dentinal tubules. By simulating the developmental microenvironment, multifunctional DPSC-Od-Exos demonstrated promising potential for reconstructing dentin-like tissue, vascular networks, and neural architectures, thereby enhancing our understanding of the therapeutic implications of DPSC-Od-Exos in regenerative endodontic treatment.

Regenerating inflamed bone defects represents a severe clinical challenge due to the undesirable inflammatory microenvironment. The inflammatory stimulus poses a weighty threat to the regenerative capacity of endogenously derived mesenchymal stem cells (MSCs), which are mainly responsible for osteogenic differentiation, thereby resulting in compromised endogenous bone formation. Consequently, alleviating the biological characteristics of inflammatory-impaired MSCs is crucial for promoting inflamed bone regeneration. Nano-sized small extracellular vesicles (sEVs) have emerged as promising therapeutic tools to orchestrate MSCs fate due to their intrinsic biocompatibility and encapsulated bioactive contents. In the present study, we extracted sEVs from youthful and adult dental pulp MSCs and explored their ability to recover inflammation-compromised periodontal ligament stem cells (IPDLSCs). The results indicated that both types of sEVs were capable of facilitating IPDLSCs osteogenesis. However, young sEVs exhibited a more robust potential at a lower concentration compared to adult sEVs. Mechanically, young sEVs enhanced the expression of bone morphogenetic protein 4 (BMP4) via delivering the protein Biglycan, which correspondingly promoted the osteogenic capability of IPDLSCs. Collectively, our findings emphasized that young sEVs hold enormous potential to rescue the inherent function and regenerative competence of inflammation-impaired MSCs, shedding light on their promising therapeutic prospects for infected bone regeneration.

再生性牙髓治疗利用组织工程方法修复缺损牙本质和牙髓组织，再生具有正常生理功能的牙髓-牙本质复合体，以期恢复牙髓的血管神经化和免疫功能并重建管样牙本质。显著影响再生性牙髓治疗效能的因素包括干细胞、生物信号分子和生物支架材料。根据是否来自牙源性组织，干细胞可分为牙源性干细胞（如牙髓干细胞）和非牙源性干细胞（如骨髓间充质干细胞）。鉴于干细胞具有倾向分化为其原始来源组织的特性，牙源性干细胞在再生性牙髓治疗中展现出一定优势。联合应用多种信号分子并通过激活信号转导通路如Wnt/β-catenin、BMP/Smad，对改善干细胞迁移、增殖、成牙本质细胞分化和血管神经再生潜能发挥关键作用。适用于再生性牙髓治疗的生物支架材料包括自然来源材料和人工合成材料，人工合成材料应模仿天然组织进行生物仿生修饰，以营造适宜的再生微环境并实现对信号转导通路的时空调控。牙髓-牙本质复合体再生的真正实现有赖于干细胞移植和干细胞归巢两大策略。干细胞归巢策略因可避免干细胞的体外分离和培养而在临床操作上更具优势，但如何提高干细胞归巢成功率并促进其增殖和分化以实现牙髓-牙本质复合体真正再生仍有待解决。本文介绍了诱导牙髓-牙本质复合体再生的影响因素，探讨其科学、可行的生物学策略，并展望再生性牙髓治疗未来的研究方向，以期为再生性牙髓治疗临床转化和推广应用提供参考。