Effect of photobiomodulation therapy with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells.

This study aimed to compare the effects of photobiomodulation therapy (PBMT) with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells (PDLMSCs). In this in vitro, experimental study, PDLMSCs were obtained from the Iranian Genetic Bank and cultured in osteogenic medium. They were then subjected to irradiation of 660 and 980 nm diode lasers, and their viability was assessed after one, two, and three irradiation cycles using the methyl thiazolyl tetrazolium (MTT) assay. The cells also underwent DAPI staining, cell apoptosis assay by using the Annexin V/PI, Alizarin Red staining, and real-time polymerase chain reaction (PCR) for assessment of the expression of osteogenic genes. Data were analyzed by two-way ANOVA. The two laser groups had no significant difference in cell apoptosis according to the results of DAPI staining. Both laser groups showed higher cell viability in the MTT assay at 4 and 6 days compared with the control group. Annexin V/PI results showed higher cell viability in both laser groups at 4 days compared with the control group. Rate of early and late apoptosis was lower in both laser groups than the control group at 4 days. Necrosis had a lower frequency in 980 nm laser group than the control group on day 6. Alizarin Red staining showed higher cell differentiation in both laser groups after 3 irradiation cycles than the control group. The highest expression of osteopontin (OPN), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) was noted in 660 nm laser group with 3 irradiation cycles at 14 days, compared with the control group. PBMT with 660 and 980 nm diode lasers decreased apoptosis and significantly increased PDLMSC differentiation after 3 irradiation cycles.

Inhibition of 12/15-lipoxygenase reduces orthodontically induced root resorption in rats.

OBJECTIVES: To investigate whether the inhibition of 12/15-lipoxygenase (12/15-LOX), one of the core enzymes of the arachidonic acid cascade, suppresses orthodontically induced root resorption (OIRR), and examine the involvement of the hyaline degeneration of periodontal ligament cells and odontoclast differentiation. MATERIALS AND METHODS: The left maxillary first molars of 10-week-old male Wistar rats were moved mesially for 14 days using a closed-coil spring (25 cN) inserted between the first molar and incisor. The rats were intraperitoneally administered with a 12/15-LOX specific inhibitor (ML-351; 0.05 mmol/kg) daily in the experimental group or vehicle (dimethyl sulfoxide) in the control group. Tooth movement was measured using microcomputed tomography on day 14. The appearance of OIRR, hyaline degeneration, osteoclasts, and odontoclasts was evaluated via histological analysis. Immunohistochemical staining for receptor-activated NF-kB ligand (RANKL) and osteoprotegerin was performed. RESULTS: OIRR observed on day 14 in the control group was strongly suppressed by ML-351 treatment. Hyaline degeneration observed on the compression side on day 3 and the appearance of osteoclasts and odontoclasts on days 3 and 14 were significantly suppressed by ML-351. RANKL expression on day 3 was significantly suppressed by ML-351. These key processes in OIRR were substantially suppressed by ML-351 treatment. CONCLUSIONS: Inhibition of 12/15-LOX reduced OIRR by suppressing hyaline degeneration and subsequent odontoclast differentiation.

The cytoskeleton dynamics-dependent LINC complex in periodontal ligament stem cells transmits mechanical stress to the nuclear envelope and promotes YAP nuclear translocation.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) are important seed cells in tissue engineering and clinical applications. They are the priority receptor cells for sensing various mechanical stresses. Yes-associated protein (YAP) is a recognized mechanically sensitive transcription factor. However, the role of YAP in regulating the fate of PDLSCs under tension stress (TS) and its underlying mechanism is still unclear. METHODS: The effects of TS on the morphology and fate of PDLSCs were investigated using fluorescence staining, transmission electron microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). Then qRT-PCR, western blotting, immunofluorescence staining and gene knockdown experiments were performed to investigate the expression and distribution of YAP and its correlation with PDLSCs proliferation. The effects of cytoskeleton dynamics on YAP nuclear translocation were subsequently explored by adding cytoskeleton inhibitors. The effect of cytoskeleton dynamics on the expression of the LINC complex was proved through qRT-PCR and western blotting. After destroying the LINC complex by adenovirus, the effects of the LINC complex on YAP nuclear translocation and PDLSCs proliferation were investigated. Mitochondria-related detections were then performed to explore the role of mitochondria in YAP nuclear translocation. Finally, the in vitro results were verified by constructing orthodontic tooth movement models in Sprague-Dawley rats. RESULTS: TS enhanced the polymerization and stretching of F-actin, which upregulated the expression of the LINC complex. This further strengthened the pull on the nuclear envelope, enlarged the nuclear pore, and facilitated YAP's nuclear entry, thus enhancing the expression of proliferation-related genes. In this process, mitochondria were transported to the periphery of the nucleus along the reconstructed microtubules. They generated ATP to aid YAP's nuclear translocation and drove F-actin polymerization to a certain degree. When the LINC complex was destroyed, the nuclear translocation of YAP was inhibited, which limited PDLSCs proliferation, impeded periodontal tissue remodeling, and hindered tooth movement. CONCLUSIONS: Our study confirmed that appropriate TS could promote PDLSCs proliferation and periodontal tissue remodeling through the mechanically driven F-actin/LINC complex/YAP axis, which could provide theoretical guidance for seed cell expansion and for promoting healthy and effective tooth movement in clinical practice.

Establishing legal threshold of 18-years based on the assessment of mandibular molars using three different methods - An observational study.

BACKGROUND: The study evaluates the feasibility of employing the radiographic visibility of the root pulp and periodontal ligament in mandibular molars for age estimation, particularly focusing on the 18 years of age threshold. This study additionally investigates the potential of root canal width reduction in mandibular molars, as a reliable method for forensic age estimation in living individuals. MATERIALS AND METHODS: A cross-sectional study was conducted to assess the radiographic visibility of the root pulp (RPV) and the root canal width (RCW) of mandibular first, second, and third molars along with the radiographic visibility of the periodontal ligament (PLV) of mandibular third molars, in a sample of 403 individuals aged 16-25 years (220 males and 183 females). Data regarding age for different stages of RPV and PLV and various types of RCW were recorded and observed for sex-based differences. Results obtained were tabulated and descriptive statistics were applied to summarise the findings. RESULTS: Individuals over 18 years old were classified with higher accuracy using stage 3 of the RPV scoring system in all mandibular molars (first, second, and third) compared to stage 2, which was also effective for the second and third molars. This result held regardless of sex and side examined. Additionally, root canal width (RCW) assessment demonstrated that individuals with RCW types A, B, and C were more likely to be under 18 years old in both sexes. Conversely, individuals with RCW type U on the right side for males and the left side for females exhibited a higher likelihood of being above 18 years old. CONCLUSION: The study suggests that the assessment of mandibular molars could potentially serve as an auxiliary tool in age estimation methods, particularly for approximating individuals around the 18 years of age threshold. Further investigation is warranted to explore the potential application of root canal width measurements in forensic age estimation.

The expression of signal regulatory protein alpha (SIRPα) in periodontal cells and tissue.

Signal regulatory protein alpha (SIRPα) is mainly expressed by cells of myeloid origin. This membrane glycoprotein is shown to be involved in regulation of different inflammatory conditions, such as colitis and arthritis. However, SIRPα has not been investigated in relationship to periodontitis, an inflammatory condition affecting the tooth supporting tissues. We aim to investigate if resident cells in the periodontium express SIRPα and whether a possible expression is affected by inflammatory conditions. Primary human keratinocytes, fibroblasts, periodontal ligament cells, and osteoblasts were cultured with or without the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) or interleukin-1-beta (IL-1ß). All different periodontal cell types showed a basal mRNA expression of SIRPα. Pro-inflammatory cytokines induced a 2-3-fold significant increase in SIRPα expression in both cultured human gingival fibroblasts and osteoblasts but neither in keratinocytes nor in periodontal ligament cells. Tissue sections from human gingival tissue biopsies were histochemically stained for SIRPα. Epithelial keratinocytes and gingival fibroblasts stained positive in sections from periodontally healthy as well as in sections from periodontitis. In periodontitis sections, infiltrating leukocytes stained positive for SIRPα. We highlight our finding that oral keratinocytes, gingival fibroblasts, and periodontal ligament cells do express SIRPα, as this has not been presented before. The fact that inflammatory stimulation of gingival fibroblasts increased the expression of SIRPα, while an increased expression by gingival fibroblasts in periodontitis tissue in situ could not be detected, is indeed contradictory.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-ßI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

CB1 Promotes Osteogenic Differentiation Potential of Periodontal Ligament Stem Cells by Enhancing Mitochondrial Transfer of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVE: To reveal the role and mechanism of cannabinoid receptor 1 (CB1) and mitochondria in promoting osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the inflammatory microenvironment. METHODS: Bidirectional mitochondrial transfer was performed in bone mesenchymal stem cells (BMSCs) and PDLSCs. Laser confocal microscopy and quantitative flow cytometry were used to observe the mitochondrial transfer and quantitative mitochondrial transfer efficiency. Realtime reverse transcription polymerase chain reaction (RT-PCR) was employed to detect gene expression. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and quantitative calcium ion analysis were used to evaluate the degree of osteogenic differentiation of PDLSCs. RESULTS: Bidirectional mitochondrial transfer was observed between BMSCs and PDLSCs. The indirect co-culture system could simulate intercellular mitochondrial transfer. Compared with the conditioned medium (CM) for BMSCs, that for HA-CB1 BMSCs could significantly enhance the mineralisation ability of PDLSCs. The mineralisation ability of PDLSCs could not be enhanced after removing the mitochondria in CM for HA-CB1 BMSCs. The expression level of HO-1, PGC-1α, NRF-1, ND1 and HK2 was significantly increased in HA-CB1 BMSCs. CONCLUSION: CM for HA-CB1 BMSCs could significantly enhance the damaged osteogenic differentiation ability of PDLSCs in the inflammatory microenvironment, and the mitochondria of CM played an important role. CB1 was related to the activation of the HO-1/PGC-1α/NRF-1 mitochondrial biogenesis pathway, and significantly increased the mitochondrial content in BMSCs.

Mechanisms of mechanical force in periodontal homeostasis: a review.

Mechanical forces affect periodontal health through multiple mechanisms. Normally, mechanical forces can boost soft and hard tissue metabolism. However, excessive forces may damage the periodontium or result in irreversible inflammation, whereas absence of occlusion forces also leads to tissue atrophy and bone resorption. We systemically searched the PubMed and Web of Science databases and found certain mechanisms of mechanical forces on immune defence, extracellular matrix (ECM) metabolism, specific proteins, bone metabolism, characteristic periodontal ligament stem cells (PDLSCs) and non-coding RNAs (ncRNAs) as these factors contribute to periodontal homeostasis. The immune defence functions change under forces; genes, signalling pathways and proteinases are altered under forces to regulate ECM metabolism; several specific proteins are separately discussed due to their important functions in mechanotransduction and tissue metabolism. Functions of osteocytes, osteoblasts, and osteoclasts are activated to maintain bone homeostasis. Additionally, ncRNAs have the potential to influence gene expression and thereby, modify tissue metabolism. This review summarizes all these mechanisms of mechanical forces on periodontal homeostasis. Identifying the underlying causes, this review provides a new perspective of the mechanisms of force on periodontal health and guides for some new research directions of periodontal homeostasis.

Nanosilicates facilitate periodontal regeneration potential by activating the PI3K-AKT signaling pathway in periodontal ligament cells.

The recent development of nanobiomaterials has shed some light on the field of periodontal tissue regeneration. Laponite (LAP), an artificially synthesized two-dimensional (2D) disk-shaped nanosilicate, has garnered substantial attention in regenerative biomedical applications owing to its distinctive structure, exceptional biocompatibility and bioactivity. This study endeavors to comprehensively evaluate the influence of LAP on periodontal regeneration. The effects of LAP on periodontal ligament cells (PDLCs) on osteogenesis, cementogenesis and angiogenesis were systematically assessed, and the potential mechanism was explored through RNA sequencing. The results indicated that LAP improved osteogenic and cementogenic differentiation of PDLCs, the regulatory effects of LAP on PDLCs were closely correlated with activation of PI3K-AKT signaling pathway. Moreover, LAP enhanced angiogenesis indirectly via manipulating paracrine of PDLCs. Then, LAP was implanted into rat periodontal defect to confirm its regenerative potential. Both micro-CT and histological analysis indicated that LAP could facilitate periodontal tissue regeneration in vivo. These findings provide insights into the bioactivity and underlying mechanism of LAP on PDLCs, highlighting it might be a potential therapeutic option in periodontal therapy.

M2 macrophage-derived exosomes enable osteogenic differentiation and inhibit inflammation in human periodontal ligament stem cells through promotion of CXCL12 expression.

BACKGROUND: Periodontitis is a dental disease characterized by inflammation of periodontal tissues and loss of the periodontal ligaments and alveolar bone. Exosomes are a class of extracellular vesicles that are involved in a variety of diseases by releasing active substances. In this study, we aimed to investigate the effect and mechanism of exosomes from M2 polarized macrophages (M2-exos) on osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). METHODS: M2-exos were isolated from IL-4-induced RAW264.7 cells (M2 macrophages) and then treated on hPDLSCs. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, measurement of osteogenic differentiation-related genes and proteins, and inflammation was evaluated by measuring the levels of inflammatory factors. The mechanism of M2-exo was confirmed through qPCR, western blot, ALP and ARS staining. RESULTS: Results suggested that M2-exo improved osteogenic differentiation and inhibited inflammation in LPS-induced hPDLSCs. CXCL12 expression was elevated in M2 macrophages, but decreased in LPS-induced hPDLSCs. Moreover, the effect of M2-exo on osteogenic differentiation and inflammation in LPS-induced hPDLSCs was reversed by CXCL12 knockdown. CONCLUSION: We demonstrated that M2-exo facilitated osteogenic differentiation and suppressed inflammation in LPS-induced hPDLSCs through promotion of CXCL12 expression. These results suggested the potential of M2-exo in the treatment of periodontitis, which may provide a new theoretical basis for M2-exo treatment of periodontitis.

FoxO1-Overexpressed Small Extracellular Vesicles Derived from hPDLSCs Promote Periodontal Tissue Regeneration by Reducing Mitochondrial Dysfunction to Regulate Osteogenesis and Inflammation.

Purpose: Periodontitis is a chronic infectious disease characterized by progressive inflammation and alveolar bone loss. Forkhead box O1 (FoxO1), an important regulator, plays a crucial role in maintaining bone homeostasis and regulating macrophage energy metabolism and osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, FoxO1 was overexpressed into small extracellular vesicles (sEV) using engineering technology, and effects of FoxO1-overexpressed sEV on periodontal tissue regeneration as well as the underlying mechanisms were investigated. Methods: Human periodontal ligament stem cell (hPDLSCs)-derived sEV (hPDLSCs-sEV) were isolated using ultracentrifugation. They were then characterized using transmission electron microscopy, Nanosight, and Western blotting analyses. hPDLSCs were treated with hPDLSCs-sEV in vitro after stimulation with lipopolysaccharide, and osteogenesis was evaluated. The effect of hPDLSCs-sEV on the polarization phenotype of THP-1 macrophages was also evaluated. In addition, we measured the reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells. Experimental periodontitis was established in vivo in mice. HPDLSCs-sEV or phosphate-buffered saline (PBS) were injected into periodontal tissues for four weeks, and the maxillae were collected and assessed by micro-computed tomography, histological staining, and small animal in vivo imaging. Results: In vitro, FoxO1-overexpressed sEV promoted osteogenic differentiation of hPDLSCs in the inflammatory environment and polarized THP-1 cells from the M1 phenotype to the M2 phenotype. Furthermore, FoxO1-overexpressed sEV regulated the ROS level, ATP production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells in the inflammatory environment. In the in vivo analyses, FoxO1-overexpressed sEV effectively promoted bone formation and inhibited inflammation. Conclusion: FoxO1-overexpressed sEV can regulate osteogenesis and immunomodulation. The ability of FoxO1-overexpressed sEV to regulate inflammation and osteogenesis can pave the way for the establishment of a therapeutic approach for periodontitis.

Sclerostin antibody corrects periodontal disease in type 2 diabetic mice.

Type 2 diabetes (T2D) is on the rise worldwide and is associated with various complications in the oral cavity. Using an adult-onset diabetes preclinical model, we demonstrated profound periodontal alterations in T2D mice, including inflamed gingiva, disintegrated periodontal ligaments (PDLs), marked alveolar bone loss, and unbalanced bone remodeling due to decreased formation and increased resorption. Notably, we observed elevated levels of the Wnt signaling inhibitor sclerostin in the alveolar bone of T2D mice. Motivated by these findings, we investigated whether a sclerostin-neutralizing antibody (Scl-Ab) could rescue the compromised periodontium in T2D mice. Administering Scl-Ab subcutaneously once a week for 4 weeks, starting 4 weeks after T2D induction, led to substantial increases in bone mass. This effect was attributed to the inhibition of osteoclasts and promotion of osteoblasts in both control and T2D mice, effectively reversing the bone loss caused by T2D. Furthermore, Scl-Ab stimulated PDL cell proliferation, partially restored the PDL fibers, and mitigated inflammation in the periodontium. Our study thus established a T2D-induced periodontitis mouse model characterized by inflammation and tissue degeneration. Scl-Ab emerged as a promising intervention to counteract the detrimental effects of T2D on the periodontium, exhibiting limited side effects on other craniofacial hard tissues.

KAT6A/YAP/TEAD4 pathway modulates osteoclastogenesis by regulating the RANKL/OPG ratio on the compression side during orthodontic tooth movement.

BACKGROUND: Orthodontic tooth movement (OTM) is a dynamic equilibrium of bone remodeling, involving the osteogenesis of new bone and the osteoclastogenesis of old bone, which is mediated by mechanical force. Periodontal ligament stem cells (PDLCSs) in the periodontal ligament (PDL) space can transmit mechanical signals and regulate osteoclastogenesis during OTM. KAT6A is a histone acetyltransferase that plays a part in the differentiation of stem cells. However, whether KAT6A is involved in the regulation of osteoclastogenesis by PDLSCs remains unclear. RESULTS: In this study, we used the force-induced OTM model and observed that KAT6A was increased on the compression side of PDL during OTM, and also increased in PDLSCs under compression force in vitro. Repression of KAT6A by WM1119, a KAT6A inhibitor, markedly decreased the distance of OTM. Knockdown of KAT6A in PDLSCs decreased the RANKL/OPG ratio and osteoclastogenesis of THP-1. Mechanistically, KAT6A promoted osteoclastogenesis by binding and acetylating YAP, simultaneously regulating the YAP/TEAD axis and increasing the RANKL/OPG ratio in PDLSCs. TED-347, a YAP-TEAD4 interaction inhibitor, partly attenuated the elevation of the RANKL/OPG ratio induced by mechanical force. CONCLUSION: Our study showed that the PDLSCs modulated osteoclastogenesis and increased the RANKL/OPG ratio under mechanical force through the KAT6A/YAP/TEAD4 pathway. KAT6A might be a novel target to accelerate OTM.

Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells.

BACKGROUND: The role of periodontal ligament stem cells (PDLSCs) in repairing periodontal destruction is crucial, but their functions can be impaired by excessive oxidative stress (OS). Nocardamine (NOCA), a cyclic siderophore, has been shown to possess anti-cancer and anti-bacterial properties. This study aimed to investigate the protective mechanisms of NOCA against OS-induced cellular dysfunction in PDLSCs. METHODS: The cytotoxicity of NOCA on PDLSCs was assessed using a CCK-8 assay. PDLSCs were then treated with hydrogen peroxide (H2O2) to induce OS. ROS levels, cell viability, and antioxidant factor expression were analyzed using relevant kits after treatment. Small molecule inhibitors U0126 and XAV-939 were employed to block ERK signaling and Wnt pathways respectively. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining of mineralized nodules. Expression levels of osteogenic gene markers and ERK pathway were determined via real-time quantitative polymerase chain reaction (RT-qPCR) or western blot (WB) analysis. ß-catenin nuclear localization was examined by western blotting and confocal microscopy. RESULTS: NOCA exhibited no significant cytotoxicity at concentrations below 20 µM and effectively inhibited H2O2-induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H2O2-stimulated PDLSCs. Mechanistically, NOCA increased p-ERK level and promoted ß-catenin translocation into the nucleus; however, blocking ERK pathway disrupted the osteogenic protection provided by NOCA and impaired its ability to induce ß-catenin nuclear translocation under OS conditions in PDLSCs. CONCLUSIONS: NOCA protected PDLSCs against H2O2-induced OS and effectively restored impaired osteogenic differentiation in PDLSCs by modulating the ERK/Wnt signaling pathway.

Herpes Simplex Virus Type 1 Infection of Human Periodontal Ligament.

The periodontal ligament (PDL) is a complex connective tissue that connects the tooth root to the dental alveolar bone and plays crucial mechanical roles. PDL also exhibits regenerative roles and regulatory functions to maintain periodontium integrity and homeostasis. While PDL exposure to oral microbial pathogens is common, virtually nothing is known regarding viral infections of PDL. In particular, human herpes simplex virus type 1 (HSV-1) persistently infects the oral cavity through infections of the oral epithelium, connective tissue and neurons. While the oral spread of HSV-1 is generally asymptomatic, this virus has also been implicated in various oral pathologies. In this study, using a primary cell model derived from PDL (PDL cells), and whole surgical fragments of PDL, we provide evidence supporting the efficient infection of PDL by HSV-1 and the promotion of cytopathic effects. Infection of PDL by HSV-1 was also associated with an acute innate inflammatory response, as illustrated by the production of antiviral interferons and pro-inflammatory cytokines. Furthermore, this inflammatory response to HSV-1 was exacerbated in the presence of bacterial-derived products, such as peptidoglycans. This work therefore highlights the ability of HSV-1 to infect mesenchymal cells from PDL, suggesting that PDL may serve as a viral reservoir for the periodontal spread of HSV-1. Moreover, this raises questions about HSV-1 oral pathogenesis, as HSV-1-associated cytopathic and inflammatory effects may contribute to profound alterations of PDL integrity and functioning.

Age-related mitophagy regulates orthodontic tooth movement by affecting PDLSCs mitochondrial function and RANKL/OPG.

A thorough comprehension of age-related variances in orthodontic tooth movement (OTM) and bone remodeling response to mechanical force holds significant implications for enhancing orthodontic treatment. Mitophagy plays a crucial role in bone metabolism and various age-related diseases. However, the impact of mitophagy on the bone remodeling process during OTM remains elusive. Using adolescent (6 weeks old) and adult (12 months old) rats, we established OTM models and observed that orthodontic force increased the expression of the mitophagy proteins PTEN-induced putative kinase 1 (PINK1) and Parkin, as well as the number of tartrate-resistant acid phosphatase-positive osteoclasts and osteocalcin-positive osteoblasts. These biological changes were found to be age-related. In vitro, compression force loading promoted PINK1/Parkin-dependent mitophagy in periodontal ligament stem cells (PDLSCs) derived from adolescents (12-16 years old) and adults (25-35 years old). Furthermore, adult PDLSCs exhibited lower levels of mitophagy, impaired mitochondrial function, and a decreased ratio of RANKL/OPG compared to young PDLSCs after compression. Transfection of siRNA confirmed that inhibition of mitophagy in PDLSC resulted in decreased mitochondrial function and reduced RANKL/OPG ratio. Application of mitophagy inducer Urolithin A enhanced bone remodeling and accelerated OTM in rats, while the mitophagy inhibitor Mdivi-1 had the opposite effect. These findings indicate that force-stimulated PDLSC mitophagy contributes to alveolar bone remodeling during OTM, and age-related impairment of mitophagy negatively impacts the PDLSC response to mechanical stimulus. Our findings enhance the understanding of mitochondrial mechanotransduction and offer new targets to tackle current clinical challenges in orthodontic therapy.

Metformin enhances the therapeutic effects of extracellular vesicles derived from human periodontal ligament stem cells on periodontitis.

Metformin has shown outstanding anti-inflammatory and osteogenic abilities. Mesenchymal stem cell-derived extracellular vesicles (EVs) reveal promising therapeutic potency by carrying various biomolecules. This study explored the effects of metformin on the therapeutic potential of EVs derived from human periodontal ligament stem cells (PDLSCs) for periodontitis. PDLSCs were cultured in osteogenic medium with or without metformin, and the supernatant was then collected separately to extract EVs and metformin-treated EVs (M-EVs). After identifying the characteristics, we evaluated the anti-inflammatory and osteogenic effects of EVs and M-EVs in vivo and in vitro. Osteogenic differentiation of PDLSCs was markedly enhanced after metformin treatment, and the effect was dramatically inhibited by GW4896, an inhibitor of EVs' secretion. Metformin significantly increased EVs' yields and improved their effects on cell proliferation, migration, and osteogenic differentiation. Moreover, metformin significantly enhanced the osteogenic ability of EVs on inflammatory PDLSCs. Animal experiments revealed that alveolar bone resorption was dramatically reduced in the EVs and M-EVs groups when compared to the periodontitis group, while the M-EVs group showed the lowest levels of alveolar bone loss. Metformin promoted the osteogenic differentiation of PDLSCs partly through EVs pathway and significantly enhanced the secretion of PDLSCs-EVs with superior pro-osteogenic and anti-inflammatory potential, thus improving EVs' therapeutic potential on periodontitis.

Youthful small extracellular vesicles restore the function and reparative capacity of inflammatory-impaired periodontal ligament stem cells via delivering protein biglycan for bone regeneration.

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.

Centrifugal Microfluidic Cell Culture Platform for Physiologically Relevant Virus Infection Studies: A Case Study with HSV-1 Infection of Periodontal Cells.

Static well plates remain the gold standard to study viral infections in vitro, but they cannot accurately mimic dynamic viral infections as they occur in the human body. Therefore, we established a dynamic cell culture platform, based on centrifugal microfluidics, to study viral infections in perfusion. To do so, we used human primary periodontal dental ligament (PDL) cells and herpes simplex virus-1 (HSV-1) as a case study. By microscopy, we confirmed that the PDL cells efficiently attached and grew in the chip. Successful dynamic viral infection of perfused PDL cells was monitored using fluorescent imaging and RT-qPCR-based experiments. Remarkably, viral infection in flow resulted in a gradient of HSV-1-infected cells gradually decreasing from the cell culture chamber entrance towards its end. The perfusion of acyclovir in the chip prevented HSV-1 spreading, demonstrating the usefulness of such a platform for monitoring the effects of antiviral drugs. In addition, the innate antiviral response of PDL cells, measured by interferon gene expression, increased significantly over time in conventional static conditions compared to the perfusion model. These results provide evidence suggesting that dynamic viral infections differ from conventional static infections, which highlights the need for more physiologically relevant in vitro models to study viral infections.

High concentrations of NaF aggravate periodontitis by promoting M1 polarization in macrophages.

High-concentration fluoride treatment is commonly used to prevent dental caries in the oral cavity, and fluorine-containing protective paint is used to alleviate common root sensitivity symptoms in patients with periodontitis after periodontal treatment. Recent studies have confirmed its safe use in normal oral environments. However, whether fluoride treatment affects the progression of periodontitis in an inflammatory microenvironment remains unclear. Immunometabolism is crucial for maintaining bone regeneration and repair in periodontitis, and the precise regulation of macrophage polarisation is crucial to this process. Fluoride can influence the immune microenvironment of bone tissue by regulating immune metabolic processes. Herein, we investigated the effects of high concentrations of sodium fluoride (NaF) on periodontal tissues. We examined the expression of osteogenic and M1/M2 macrophage polarisation markers and glucose metabolism in macrophages. RNA sequencing was used to study differentially expressed genes related to M1 polarisation and glucose metabolism in treated macrophages. The results showed that NaF indirectly affects human periodontal ligament cells (hPDLCs), aggravating bone loss, tissue destruction, and submandibular lymph node drainage. Furthermore, NaF promoted glycolysis in macrophages and M1 polarisation while inhibiting osteogenic differentiation. These findings suggest that NaF has a direct effect on hPDLCs. Moreover, we found that high concentrations of NaF stimulated M1 polarisation in macrophages by promoting glycolysis. Overall, these results suggest that M1 macrophages promote the osteoclastic ability of hPDLCs and inhibit their osteogenic ability, eventually aggravating periodontitis. These findings provide important insights into the mechanism of action of NaF in periodontal tissue regeneration and reconstruction, which is critical for providing appropriate recommendations for the use of fluoride in patients with periodontitis.