Dental implants are currently one of the primary methods for restoring tooth loss, and osseointegration is the criterion that determines implant success[43]. However, occlusal trauma and alveolar bone resorption can readily occur following implant restoration due to the absence of PDL tissue and periodontal proprioceptors[44]. Bio-root formation based on tissue-engineered stem cell/scaffold materials is projected to replace implants as a revolutionary treatment for tooth loss [4]. DFCs have been identified as viable candidate seed cells for the purpose of bio-root construction[45]. As precursor cells of periodontal tissue, DFCs can form periodontium, cementum, and alveolar bone and exhibit osteogenic, neurogenic, and lipogenic differentiation under specific conditions[46, 47]. In addition to precursor cells, a suitable scaffold with properties and anatomical structure comparable to the natural tooth is required. According to several studies, porcine tooth-derived dentin matrix, a type of xECM with a histological structure and bioactive factors comparable to those in human-derived dentin matrix, can promote odontogenesis and biomineralization[8]. xECM composite odontogenic stem cells have been widely used to construct bio-roots while regenerating dentin structure, dental pulp-like tissue, PDL, and proprioceptors for physiologically plausible functional healing[9, 10]. However, unfavorable oxidative stress is a substantial barrier to the success of xECM-based organ transplantation, raising the need for antioxidant capacity in xenograft regeneration[48, 49].
Oxidative stress is a phenomenon that arises from the disturbance of redox homeostasis, wherein there is a transient or chronic elevation in ROS levels or a reduction in the capacity to scavenge ROS. This condition leads to significant cellular and tissue damage[50]. xECM-based bio-roots exhibited aberrant oxidative stress and inflammation owing to immunological rejection, as seen in a previous study conducted with implanted xenografts[51]. Once the graft has been implanted, a significant number of inflammatory cells gather around it, resulting in a substantial accumulation of ROS and inflammation[15]. These inflammatory factors and ROS increase the number of inflammatory cells at the graft site while also generating copious amounts of superoxide anion and H2O2 [16]. This cycle results in considerable ROS generation, which directly causes oxidative damage to DNA, proteins, and lipids and pathological changes to the tissue[52, 53]. To further detect the level of oxidative stress in cells, we can detect the expression of 8-OHdG, 3-NT, and MDA, which are biomarkers for DNA oxidative damage[54], protein oxidation[55], and lipid oxidative damage, respectively[56]. Here, the significantly increased expression of these biomarkers confirmed that xECM and H2O2 induced oxidative damage to seed cells.
EVs refer to nanoparticles that are enclosed within naturally derived membranes and are released by various types of cells. Additionally, they play a pivotal role in facilitating intercellular communication by transporting proteins, lipids, nucleic acids, and organelles to adjacent or remote cells[57, 58]. EVs perform both physiological and pathogenic functions, which makes them attractive therapeutic targets[59, 60]. Consistent with a previous report[61, 62], we found that hASC-EVs were absorbed by DFCs via the endocytic pathway and that they significantly improved cell function. MSC-EVs can protect cellular functions against oxidative stress-induced injury [25]. Xian et al. provided evidence that MSC-EVs have a protective effect on endothelial cells, shielding them from senescence induced by oxidative stress. Additionally, the researchers observed that MSC-EVs enhanced the migration of senescent cells and facilitated the restoration of tube formation[63]. In the present study, we analyzed numerous functions to assess whether hASC-EVs promote the functional recovery of oxidative stress. The experimental results from proliferation, migration, and apoptosis assays provide evidence that the administration of hASC-EVs resulted in enhanced cellular activity, increased cell proliferation, facilitated cell migration, and decreased the occurrence of apoptotic cells in the presence of oxidative stress. Overall, EV treatment considerably improved and enhanced cellular function. Additionally, EVs can regulate the effects of cellular oxidative stress, including seizure-induced neuronal damage and UV-induced epidermis damage [61, 64]. However, the efficacy of EVs against xenogeneic bio-root-induced oxidative stress is inadequately defined. In the present study, the significantly increased ROS generation and oxidative damage marker expression confirmed that xECM and H2O2 induced oxidative damage to seed cells. However, these effects were reversed by hASC-EV treatment. These results verified that EVs may regulate the oxidative stress levels of cells to a certain extent and inhibit oxidative stress-induced injury in xenogeneic bio-roots.
Mitochondria are one of the target organelles for oxidative damage[65]. As they are the center of cellular homeostasis, any damage to mitochondria disrupts intracellular metabolism and reduces the supply of ATP, which exacerbates cell damage and induces apoptosis[66, 67]. Several studies have shown that certain miRNAs in EVs, including miR-30 (ref. [68]) and miR-200a-3p[69], can act directly on mitochondria to regulate mitochondrial dysfunction[70]. Consistent with these findings, we found that hASC-EVs altered mitochondrial membrane potential via the intrinsic pathway. Through the evaluation of biomarkers and cellular antioxidant enzymes[71], we assessed the antioxidant capacity of DFCs. FRAP indicates the overall levels of antioxidants[72]. SODs are the primary enzymes that catalyze the conversion of superoxide anions to H2O2 (ref. [73]), while CAT and GSH-PXs further reduce H2O2 to water. GSH-PX conversion requires the coupled oxidation of GSH to glutathione disulfide[74]. Oxidative stress lowers the cellular concentrations of FRAP, GSH-PX, CAT, and SOD. Notably, our findings revealed that hASC-EV treatment elevated the levels of these markers compared to their levels in cells under oxidative stress, demonstrating the antioxidant properties of hASC-EVs in oxidative DFCs. These results verified that hASC-EVs can regulate the oxidative stress levels of cells to a certain extent.
In addition to cellular functions and oxidative stress levels, the differentiation capacity of DFCs plays a significant role in xenobiotic tooth root regeneration, which is directly affected by tissue regeneration. Multiple studies have demonstrated that MSC-EVs possess the ability to facilitate cellular differentiation and tissue regeneration to a limited degree, while concurrently modulating the oxidative stress milieu[75]. Consistent with previous research[76, 77], excessive ROS levels inhibited the ability of MSCs to differentiate multi-directionally. Under oxidative stress, we discovered that hASC-EV treatment improved the osteogenic differentiation of DFCs by promoting mineralized nodule formation, ALP activity, and COL1 and RUNX2 levels. ALP activity and COL1 levels can be used to evaluate early osteogenic differentiation[78, 79]. The formation of mineralized nodules, which can be identified using ARS staining, is indicative of late-phase osteogenic differentiation [78]. These results demonstrate that EVs can effectively enhance the osteogenic differentiation potential of cells, even in the presence of oxidative stress, bringing it close to the levels observed under normal conditions. Furthermore, we investigated the expression of odontogenic markers in addition to the modulation of osteogenic differentiation. This effect is achieved through the upregulation of key genes involved in odontogenesis, namely DSPP, DMP-1, and periostin. DSPP and DMP-1 serve as reliable indicators of newly synthesized dentin, while periostin and COL1 are distinct markers specific to PDL tissue[80]. Our in vivo and in vitro experiments provided additional evidence to support the notion that hASC-EVs influenced osteogenesis and played a role in the formation of dentin, specifically the predentin and the odontoblast layer, as well as the PDL tissues, particularly in the presence of oxidative stress conditions. In general, the experimental data we have obtained suggest that hASC-EVs have a significant impact on the regeneration of foreign root tissues and the differentiation of DFCs into odontogenic cells. This effect has been observed both in in vivo and in vitro.
NRF2/HO-1 signaling plays a crucial role in maintaining cellular redox homeostasis under conditions of oxidative stress[81]. The findings of our study indicated that hASC-EVs exerted a protective effect against oxidative stress-induced damage to the cellular functions of seed cells and tissue regeneration. This protective mechanism was mediated through the activation of the NRF2/ HO-1 pathway, which was involved in anti-oxidation processes[82, 83]. In the cellular model of oxidative stress induced by H2O2, prior exposure to hASC-EVs expedited the degradation of KEAP1, leading to the translocation of NRF2 and improved the expression of HO-1. This was subsequently accompanied by a decline in oxidative damage[84]. The activation of the NRF2 signaling pathway in DFCs had the potential to decrease the production of ROS, enhance the activity of antioxidant enzymes, and alleviate the negative effects of elevated ROS on osteogenic and odontogenic differentiation. Multiple signaling pathways, such as PI3K/Akt, AMPK, and MAPK, have been observed to induce the activation of NRF2 in different disease models. The cellular defense against oxidative injury can be facilitated through the interplay of the PI3K/Akt and NRF2/HO-1 signaling pathways[85, 86]. It has been verified that in line with the observed NRF2 activation pattern, the administration of hASC-EVs also lead to an elevation in the phosphorylation of PI3K/Akt. This suggested that both the NRF2/HO-1 pathway and the upstream PI3K/Akt pathway played a critical role in mediating the antioxidative impact of hASC-EVs.
NRF2, as a crucial transcription factor, can act against oxidative stress by activating numerous genes encoding cytoprotective and antioxidative enzymes[87–89]. Conversely, the inhibition of NRF2 through the use of siNRF2 may attenuate these protective effects [37, 90–92]. Subsequent investigations have demonstrated that the administration of hASC-EVs did not induce any significant changes in the protein expressions of NRF2 and HO-1, as well as the differentiation processes related to bone and tooth formation, when subjected to oxidative stress in si-NRF2 DFCs. These findings suggest that the therapeutic application of hASC-EVs is unable to alleviate the oxidative harm resulting from the suppression of NRF2 in DFCs. The findings of this study indicate that hASC-EVs have the potential to modulate oxidative stress in xenobiotic tooth roots through the activation of the NRF2/HO-1 signaling pathway.
In summary, we showed that hASC-EVs could serve as a potential nanotherapeutic agent for improving the microenvironment for xenogeneic bio-root grafting and a viable strategy for PDL-like tissue regeneration. In addition, hASC-EVs may protect the biological characteristics of DFCs by maintaining cell viability, promoting migration, reducing apoptosis, eliminating cellular ROS, reducing mitochondrial change and DNA damage, enhancing cellular capacities, and enhancing antioxidant capacities to protect against redox imbalances following exposure to unfavorable transplant microenvironments via the PI3K/Akt/NRF2 defense system.