Effect of human periodontal ligament stem cell-derived exosomes on cementoblast activity.

OBJECTIVES: Exosomes derived from stem cells are a potential cell-free tool for tissue regeneration with therapeutic potential. However, its application in cementum repair is unclear. This study aimed to investigate the effect of human periodontal ligament stem cell-derived exosomes on the biological activity of cementoblasts, the main effector cells in cementum synthesis. MATERIALS AND METHODS: OCCM-30 cementoblasts were cultured with various human periodontal ligament stem cell-derived exosome concentrations. OCCM-30 cells proliferation, migration, and cementogenic mineralization were examined, along with the gene and protein expression of factors associated with cementoblastic mineralization. RESULTS: Exosomal promoted the migration, proliferation, and mineralization of OCCM-30 cells. The exosome-treated group significantly increased the expression of cementogenic-related genes and proteins. Furthermore, the expression of p-PI3K and p-AKT was enhanced by exosome administration. Treatment with a PI3K/AKT inhibitor markedly attenuated the gene and protein expression of cementoblastic factors, and this effect was partially reversed by exosome administration. CONCLUSIONS: Human periodontal ligament stem cell-derived exosomes can promote the activity of cementoblasts via the PI3K/AKT signaling pathway, providing a scientific basis for promoting the repair process in orthodontically induced inflammatory root resorption.

Sulfoconjugation of protein peptides and glycoproteins in physiology and diseases.

Protein sulfoconjugation, or sulfation, represents a critical post-translational modification (PTM) process that involves the attachment of sulfate groups to various positions of substrates within the protein peptides or glycoproteins. This process plays a dynamic and complex role in many physiological and pathological processes. Here, we summarize the importance of sulfation in the fields of oncology, virology, drug-induced liver injury (DILI), inflammatory bowel disease (IBD), and atherosclerosis. In oncology, sulfation is involved in tumor initiation, progression, and migration. In virology, sulfation influences viral entry, replication, and host immune response. In DILI, sulfation is associated with the incidence of DILI, where altered sulfation affects drug metabolism and toxicity. In IBD, dysregulation of sulfation compromises mucosal barrier and immune response. In atherosclerosis, sulfation influences the development of atherosclerosis by modulating the accumulation of lipoprotein, and the inflammation, proliferation, and migration of smooth muscle cells. The current review underscores the importance of further research to unravel the underlying mechanisms and therapeutic potential of targeting sulfoconjugation in various diseases. A better understanding of sulfation could facilitate the emergence of innovative diagnostic or therapeutic strategies.

Pregnenolone 16α-carbonitrile negatively regulates hippocampal cytochrome P450 enzymes and ameliorates phenytoin-induced hippocampal neurotoxicity.

The central nervous system is susceptible to the modulation of various neurophysiological processes by the cytochrome P450 enzyme (CYP), which plays a crucial role in the metabolism of neurosteroids. The antiepileptic drug phenytoin (PHT) has been observed to induce neuronal side effects in patients, which could be attributed to its induction of CYP expression and testosterone (TES) metabolism in the hippocampus. While pregnane X receptor (PXR) is widely known for its regulatory function of CYPs in the liver, we have discovered that the treatment of mice with pregnenolone 16α-carbonitrile (PCN), a PXR agonist, has differential effects on CYP expression in the liver and hippocampus. Specifically, the PCN treatment resulted in the induction of cytochrome P450, family 3, subfamily a, polypeptide 11 (CYP3A11), and CYP2B10 expression in the liver, while suppressing their expression in the hippocampus. Functionally, the PCN treatment protected mice from PHT-induced hippocampal nerve injury, which was accompanied by the inhibition of TES metabolism in the hippocampus. Mechanistically, we found that the inhibition of hippocampal CYP expression and attenuation of PHT-induced neurotoxicity by PCN were glucocorticoid receptor dependent, rather than PXR independent, as demonstrated by genetic and pharmacological models. In conclusion, our study provides evidence that PCN can negatively regulate hippocampal CYP expression and attenuate PHT-induced hippocampal neurotoxicity independently of PXR. Our findings suggest that glucocorticoids may be a potential therapeutic strategy for managing the neuronal side effects of PHT.

Injectable periodontal ligament stem cell-metformin-calcium phosphate scaffold for bone regeneration and vascularization in rats.

OBJECTIVES: Injectable and self-setting calcium phosphate cement scaffold (CPC) capable of encapsulating and delivering stem cells and bioactive agents would be highly beneficial for dental and craniofacial repairs. The objectives of this study were to: (1) develop a novel injectable CPC scaffold encapsulating human periodontal ligament stem cells (hPDLSCs) and metformin (Met) for bone engineering; (2) test bone regeneration efficacy in vitro and in vivo. METHODS: hPDLSCs were encapsulated in degradable alginate fibers, which were then mixed into CPC paste. Five groups were tested: (1) CPC control; (2) CPC + hPDLSC-fibers + 0% Met (CPC + hPDLSCs + 0%Met); (3) CPC + hPDLSC-fibers + 0.1% Met (CPC + hPDLSCs + 0.1%Met); (4) CPC + hPDLSC-fibers + 0.2% Met (CPC + hPDLSCs + 0.2%Met); (5) CPC + hPDLSC-fibers + 0.4% Met (CPC + hPDLSCs + 0.4%Met). The injectability, mechanical properties, metformin release, and hPDLSC osteogenic differentiation and bone mineral were determined in vitro. A rat cranial defect model was used to evaluate new bone formation. RESULTS: The novel construct had good injectability and physical properties. Alginate fibers degraded in 7 days and released hPDLSCs, with 5-fold increase of proliferation (p＜0.05). The ALP activity and mineral synthesis of hPDLSCs were increased by Met delivery (p＜0.05). Among all groups, CPC+hPDLSCs+ 0.1%Met showed the greatest cell mineralization and osteogenesis, which were 1.5-10 folds those without Met (p＜0.05). Compared to CPC control, CPC+hPDLSCs+ 0.1%Met enhanced bone regeneration in rats by 9 folds, and increased vascularization by 3 folds (p＜0.05). CONCLUSIONS: The novel injectable construct with hPDLSC and Met encapsulation demonstrated excellent efficacy for bone regeneration and vascularization in vivo in an animal model. CPC+hPDLSCs+ 0.1%Met is highly promising for dental and craniofacial applications.

Higher yield and enhanced therapeutic effects of exosomes derived from MSCs in hydrogel-assisted 3D culture system for bone regeneration.

Exosomes are emerging in tissue engineering as up-and-coming acellular therapeutics, circumventing common restrictions inherent to cell-based therapies. The characteristics and function of exosomes are affected by the bidirectional communication of their original cells and the local microenvironment in which the cells reside (e.g., the stem cell niche). However, mesenchymal stem cells (MSCs) are customarily cultured in a traditional two-dimensional monolayer, with mechanical microenvironments varying substantially in physiological one. Few reports have addressed the effects of the 3D microenvironment on exosomal osteoinductivity. Herein, a 3D culture model is engineered through collagen hydrogel. Exosomes derived from three-dimensional culture (3D-Exos) and the conventional monolayer culture (2D-Exos) are collected and compared. The 3D culture resulted in high yield exosomes and greatly improved the efficiency of exosomes collection. The in vitro results demonstrated that the 3D-Exos induced significant promotions in osteogenic gene and protein expression (e.g., Runx2, OCN, OPN, COL1A1, and ALP), proliferation, and migration of Human bone marrow mesenchymal stem cells (hBMSCs) and inhibited hBMSCs apoptosis. Importantly, mechanistic studies revealed that the upregulation of the YAP signaling pathway is the underlying mechanism. Moreover, the 3D-Exos resulted in enhanced new bone formation and Runx2/OPN activation in rats with alveolar bone defects. These findings proposed a novel idea of the 3D culture strategy used to enhance the osteoinductivity of MSC-derived exosomes. This study also provided valuable references for exosome-based clinical applications for the treatment and regeneration of tissue defects from the perspective of culture dimensions.

Periodontal ligament fibroblast-derived exosomes induced by compressive force promote macrophage M1 polarization via Yes-associated protein.

OBJECTIVES: This study aimed to investigate the biological roles and mechanisms of compressive force-stimulated periodontal ligament fibroblasts (PDLFs) on polarization of macrophages DESIGN: PDLFs were stimulated with or without static compressive force, and then conditioned medium, high-molecular weight proteins and low-molecular weight proteins were collected to treat THP-1 macrophages. RT-qPCR and flow cytometric analysis were used to evaluate the polarization of macrophages. Exosomes were isolated by ultracentrifugation method and identified via transmission electron microscopy, western-blot and nano-tracking analysis. The protein level of Yes-Associated Protein (YAP) contained in exosomes was detected by western blot. GW4869 and Verteporfin were used to inhibit exosome secretion and YAP- TEA domain transcription factor (TEAD) interaction respectively. RESULTS: Exosomes were successfully purified from PDLFs and could be efficiently incorporated into THP-1 macrophages. conditioned medium, HMW proteins and exosomes derived from compressive force-treated PDLFs significantly induce M1 polarization of macrophages. While inhibiting exosomes secretion by GW4869 treatment eliminated the inductive effect. YAP target genes, connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) were upregulated in macrophages when treated with exosomes derived from compressive force-treated PDLFs (F-Exo). YAP level was elevated in the F-Exo. When macrophages were treated with Verteporfin, expression of YAP target genes and M1 polarization were significantly downregulated. CONCLUSION: These results suggested that exosomes derived from compressive force-treated PDLFs promoted the M1 polarization of the THP-1 macrophages. The elevated level of YAP in the exosomes may be a critical factor for this response.