The potential for clinical application of MSCs in treating periodontal disease is well-established. Several clinical studies have demonstrated that autologous MSCs can enhance relevant clinical indicators in patients with periodontal disease.[28, 29] However, the optimal selection and appropriate timing for no-autologous MSCs application remain unresolved issues. Effective and precise use of MSCs requires a thorough understanding of their functional mechanisms. Previous research on MSC mechanisms in periodontal regeneration has predominantly been results-oriented.[13, 30, 31, 32] Specific mechanisms of implanted no-autologous MSCs are still poorly understood. Given the limited survival time[9, 18] of most implanted MSCs and the potential for missing crucial information in conclusion-oriented mechanism studies, this research successfully engineered DFSC lines that enable in vivo tracking of cells, facilitating the visualization of implanted cells and their derivatives. Furthermore, the efficacy of this cell line in periodontal regeneration and immune regulation was verified both in vivo and in vitro. After implanting DFSCs into periodontal defects in mice, not only the early-stage tracks between DFSCs and innate immunity system were investigated, but also the relevant immune influences were observed at the late stage of periodontal regeneration.
In vitro studies have shown that the proliferation capacity of DFSCs decreases following lentivirus infection, consistent with earlier reports that lentiviral infection can reduce cell viability and proliferation.[33, 34] Despite this reduction in proliferation, differentiation experiment and co-culture experiments demonstrate that the ability of infected DFSCs on multi-differentiation and promotion of PDLCs migration and M2 polarization remains robust. This discrepancy between cell proliferation and functional capabilities suggests that a decline in proliferation does not necessarily imply a reduction in other cellular functions. Furthermore, the immunomodulatory abilities of DFSCs and their capacity to mobilize indigenous cells may be more critical than their proliferative capacity for periodontal regeneration. This is evidenced by the observation that the repair of alveolar bone and periodontal ligaments in mice was not compromised by the diminished proliferative ability of infected DFSCs.
A detailed comparison revealed that E-DFSCs have a superior performance than DFSCs in osteogenic differentiation, promoting M2 polarization and PDLCs migration, and up-regulating COL-1 expression. In previous studies, gene overexpression was used to optimize some properties of MSCs[35, 36], and the overexpression of ACTB and CD63 in DFSCs may potentially enhance the functionality of DFSCs. Beta-actin (ACTB), a highly conserved cytoskeletal structural protein, plays critical roles in cell growth and migration[37], and its ablation can modify the ratio of globular actin (G-actin) to filamentous actin (F-actin) [38], which is essential for osteogenic and adipogenic differentiation of MSCs[39]. CD63, a member of the tetraspanin superfamily, is abundantly expressed and localized to the intraluminal vesicles of late endosomes and multivesicular bodies in EVs. [40] As a common scaffold protein for target protein loading in EVs[41], CD63 enhances EV secretion[42] and supports various biological functions, such as ferritin secretion[43] and increased biogenesis[44], thereby facilitating the exchange of information between cells. OF course, the further experiments will be planned to determine whether the overexpression of ACTB and CD63 can effectively optimize the specific functionality of DFSCs.
Preliminary in vivo tracing results indicated no visible proliferation of DFSCs after implantation; rather, the number of structurally intact DFSCs gradually declined over time in the defect area, aligning with previous studies. [9, 18] The implanted DFSCs can interact with endogenous cells via direct contact or paracrine signaling, activating these endogenous cells to participate in tissue repair. Following the implantation of DFSCs, neutrophils, not macrophages[45] or lymphocytes[46] as previously suggested, were the primary endogenous cells that migrated to the injury site to interact with DFSCs and phagocytize sEVs. Neutrophils play a critical role in the onset, progression, and healing of periodontitis. [47, 48] As a primary line of immune defense, neutrophils exhibit robust chemotaxis, rapidly moving to inflammatory sites. [49] Upon activation, neutrophils release a range of substances, including processed antigens, chemokines, cytokines, and cytotoxic effector molecules like peroxide and superoxide, [50] which influence the subsequent immune responses of antigen-presenting cells, [51] macrophages, [19] and T lymphocytes. [52] Immune regulation is crucial for tissue regeneration. Given the limited survival time of implanted MSCs and the importance of early immune regulation for tissue healing, this study focused on tracing the biological behavior of DFSCs in early stage (1,3,5 days). Of course, in order to have a deeper understanding of the fate of DFSCs and their complex interactions in the whole immune system turnover process, long-term tracking of DFSCs is necessary, and we will gradually improve this huge work in subsequent experiments.
The recruitment of neutrophils is crucial for the repair of injured or inflamed tissues. Kovtun et al. [53] discovered that granulocyte colony-stimulating factor (G-CSF) significantly enhances neutrophil recruitment, thereby accelerating fracture healing; conversely, depletion of neutrophils impairs this process. Moreover, Okan et al. [54] suggested a mechanism whereby infiltrating neutrophils promote early fracture healing through the formation of an "emergency extracellular matrix (ECM)." Subsequent research has further detailed how danger-associated molecular patterns (DAMPs) released following a fracture primarily recruit neutrophils to the injury site. These chemotactic neutrophils clear the area by phagocytizing pathogens and tissue or cellular debris, and facilitate the formation of a local hematoma by interacting with platelets to form neutrophil extracellular traps (NETs).[55] Following this, neutrophils contribute to the reconstruction of the local extracellular matrix and the establishment of a vascular network, enhancing fracture healing.[56] In our study, implanted DFSCs significantly increased neutrophil recruitment in the early stages of periodontal injury, enhancing the clearance of damaged tissues or cells and the establishment of an extracellular matrix and vascular network, thereby promoting the healing of periodontal defects.
In addition to the effective infiltration of neutrophils, the transformation of the neutrophil phenotype in the injured area is also important for the tissue healing. In the study on the subtype classification of neutrophils, it was demonstrated that different environmental conditions induce distinct neutrophil subtypes, such as N1 and N2.[57] N1 neutrophils exhibit cytotoxicity, high migration, phagocytosis, oxidative bursts, and pro-inflammatory properties but lack anti-tumor and inhibitory capabilities. Conversely, N2 neutrophils are characterized by longevity, inhibitory, anti-inflammatory, and pro-tumor properties, without exhibiting cytotoxicity, migration, or phagocytic activities.[58] In research by Yonggang[59] on myocardial infarction (MI), N1 neutrophils are the predominant phenotype in the early stages of MI (comprising over 80% of total neutrophils) due to DAMPs and the inflammatory environment. Over time post-MI, the proportion of N2 neutrophils increases, underscoring their role in resolving inflammation and aiding wound repair. Similarly, in this study, we found that over time, the phenotypic expression of N1 neutrophils gradually decreased, while the N2 type gradually increased, indicating that the transformation of the N1 and N2 phenotypes of neutrophils matched the healing process of tissues. Importantly, the promotion of N2 phenotype transformation by DFSCs can accelerate the entire immune balance enters the anti-inflammatory and repair stage earlier and faster.
As mentioned earlier, as a primary line of immune defense, the neutrophils’ phenotype transformation affects the subsequent immune response and process of tissue healing to a certain extent. Andreea's study[19] demonstrated that mediators released by neutrophil subtypes significantly influence the phenotypic modulation of macrophages. Factors released by N1 neutrophils induce macrophages to polarize towards the M1 phenotype, while exposure to N2 secretome prompts macrophages to adopt a pro-healing M2 phenotype. In this study, after DFSCs implantation, we observed the up-regulation both of N2 neutrophils in early stage of injury and M2 macrophages in late stage of regeneration. However, given the few infiltrations of macrophages in the early stage, the effect of DFSCs on M2 polarization is likely to be due to the DFSCs promote the N2 neutrophils conversion make N2 secretome increase to polarize the macrophages to M2. Of course, we need to design more accurate experiments to clarify whether there is a necessary relationship between N2 and M2 in the process of periodontal defect repair.
In addition, Wagoner[32] previously proposed the “immune clearance mechanism of dying MSCs in vivo,” wherein MSCs rapidly perish post-implantation and are phagocytized by immune cells. This clearance of dying MSCs can polarize them towards an anti-inflammatory, tolerant, and regenerative phenotype, thereby modulating the immune system and facilitating tissue repair. However, this theory has been met with skepticism, as evidence suggests that the efficacy of MSCs may rely heavily on their cellular activity and adaptability.[60, 61] In our study, immune clearance occurs not only for deceased MSCs but also for xenogeneic, implanted, surviving DFSCs. The clearance of DFSCs-derived EVs and cell debris may also promote innate immune cells, such as neutrophils and macrophages, polarizing to anti-inflammatory phenotypes. The up-regulation of N2 and M2 phenotype shifted the immune balance toward tissue healing. Therefore, the ratio of OPG/RANKL was up-regulated and osteoclast was reduced at 4th week, thereby the bone immune balance tilts toward bone-formation to promote alveolar bone regeneration.
In the immune system, the interactions and relationships among various immune cells are complex. Understanding the differential effects of MSCs on immune cells is crucial. This knowledge is essential for effectively utilizing these cells to enhance immune balance and facilitate tissue repair and regeneration at the appropriate time.