Pyroptotic macrophages induce disruption of glutamate metabolism in periodontal ligament stem cells contributing to their compromised osteogenic potential.

Macrophage pyroptosis is of key importance to host defence against pathogen infections and may participate in the progression and recovery of periodontitis. However, the role of pyroptotic macrophages in regulating periodontal ligament stem cells (PDLSCs), the main cell source for periodontium renewal, remains unclear. First, we found that macrophage pyroptosis were enriched in gingiva tissues from periodontitis patients compared with those of healthy people through immunofluorescence. Then the effects of pyroptotic macrophages on the PDLSC osteogenic differentiation were investigated in a conditioned medium (CM)-based coculture system in vitro. CM derived from pyroptotic macrophages inhibited the osteogenic differentiation-related gene and protein levels, ALP activity and mineralized nodule formation of PDLSCs. The osteogenic inhibition of CM was alleviated when pyroptosis was inhibited by VX765. Further, untargeted metabolomics showed that glutamate limitation may be the underlying mechanism. However, exogenous glutamate supplementation aggravated the CM-inhibited osteogenic differentiation of PDLSCs. Moreover, CM increased extracellular glutamate and decreased intracellular glutamate levels of PDLSCs, and enhanced the gene and protein expression levels of system xc - (a cystine/glutamate antiporter). After adding cystine to CM-based incubation, the compromised osteogenic potency of PDLSCs was rescued. Our data suggest that macrophage pyroptosis is related to the inflammatory lesions of periodontitis. Either pharmacological inhibition of macrophage pyroptosis or nutritional supplements to PDLSCs, can rescue the compromised osteogenic potency caused by pyroptotic macrophages.

Harnessing Mechanical Stress with Viscoelastic Biomaterials for Periodontal Ligament Regeneration.

The viscoelasticity of mechanically sensitive tissues such as periodontal ligaments (PDLs) is key in maintaining mechanical homeostasis. Unfortunately, PDLs easily lose viscoelasticity (e.g., stress relaxation) during periodontitis or dental trauma, which disrupt cell-extracellular matrix (ECM) interactions and accelerates tissue damage. Here, Pluronic F127 diacrylate (F127DA) hydrogels with PDL-matched stress relaxation rates and high elastic moduli are developed. The hydrogel viscoelasticity is modulated without chemical cross-linking by controlling precursor concentrations. Under cytomechanical loading, F127DA hydrogels with fast relaxation rates significantly improved the fibrogenic differentiation potential of PDL stem cells (PDLSCs), while cells cultured on F127DA hydrogels with various stress relaxation rates exhibited similar fibrogenic differentiation potentials with limited cell spreading and traction forces under static conditions. Mechanically, faster-relaxing F127DA hydrogels leveraged cytomechanical loading to activate PDLSC mechanotransduction by upregulating integrin-focal adhesion kinase pathway and thus cytoskeletal rearrangement, reinforcing cell-ECM interactions. In vivo experiments confirm that faster-relaxing F127DA hydrogels significantly promoted PDL repair and reduced abnormal healing (e.g., root resorption and ankyloses) in delayed replantation of avulsed teeth. This study firstly investigated how matrix nonlinear viscoelasticity influences the fibrogenesis of PDLSCs under mechanical stimuli, and it reveals the underlying mechanobiology, which suggests novel strategies for PDL regeneration.

Non-Impacted Third Molars: Angels or Devils?

Third molars, also known as wisdom teeth, are located in the most posterior of the tooth arch [...].

NADPH-dependent ROS accumulation contributes to the impaired osteogenic differentiation of periodontal ligament stem cells under high glucose conditions.

Diabetes mellitus is an established risk factor for periodontal disease that can aggravate the severity of periodontal inflammation and accelerate periodontal destruction. The chronic high glucose condition is a hallmark of diabetes-related pathogenesis, and has been demonstrated to impair the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), leading to delayed recovery of periodontal defects in diabetic patients. Reactive oxygen species (ROS) are small molecules that can influence cell fate determination and the direction of cell differentiation. Although excessive accumulation of ROS has been found to be associated with high glucose-induced cell damage, the underlying mechanisms remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) is an important electron donor and functions as a critical ROS scavenger in antioxidant systems. It has been identified as a key mediator of various biological processes, including energy metabolism and cell differentiation. However, whether NADPH is involved in the dysregulation of ROS and further compromise of PDLSC osteogenic differentiation under high glucose conditions is still not known. In the present study, we found that PDLSCs incubated under high glucose conditions showed impaired osteogenic differentiation, excessive ROS accumulation and increased NADPH production. Furthermore, after inhibiting the synthesis of NADPH, the osteogenic differentiation of PDLSCs was significantly enhanced, accompanied by reduced cellular ROS accumulation. Our findings demonstrated the crucial role of NADPH in regulating cellular osteogenic differentiation under high glucose conditions and suggested a new target for rescuing high glucose-induced cell dysfunction and promoting tissue regeneration in the future.

Biotin-Avidin System-Based Delivery Enhances the Therapeutic Performance of MSC-Derived Exosomes.

Exosomes (EXs) shed by mesenchymal stem cells (MSCs) are potent therapeutic agents that promote wound healing and regeneration, but when used alone in vivo, their therapeutic potency is diminished by rapid clearance and bioactivity loss. Inspired by the biotin-avidin interaction, we developed a simple yet versatile method for the immobilization of MSC-derived EXs (MSC-EXs) into hydrogels and achieved sustained release for regenerative purposes. First, biotin-modified gelatin methacryloyl (Bio-GelMA) was fabricated by grafting NHS-PEG12-biotin onto the amino groups of GelMA. Biotin-modified MSC-EXs (Bio-EXs) were then synthesized using an in situ self-assembling biotinylation strategy, which provided sufficient binding sites for MSC-EX delivery with little effect on their cargo composition. Thereafter, Bio-EXs were immobilized in Bio-GelMA via streptavidin to generate Bio-GelMA@Bio-EX hydrogels. An in vitro analysis demonstrated that Bio-EXs could be taken up by macrophages and exerted immunomodulatory effects similar to those of MSC-EXs, and Bio-GelMA@Bio-EX hydrogels provided sustained release of MSC-EXs for 7 days. After subcutaneous transplantation, a more constant retention of MSC-EXs in Bio-GelMA@Bio-EX hydrogels was observed for up to 28 days. When placed in an artificial periodontal multitissue defect, the functionalized hydrogels exhibited an optimized therapeutic performance to regrow complex periodontal tissues, including acellular cementum, periodontal ligaments (PDLs), and alveolar bone. In this context, Bio-GelMA@Bio-EX hydrogels exerted a robust immunomodulatory effect that promoted macrophage polarization toward an M2 phenotype. Our findings demonstrate that MSC-EXs delivered with the aid of the biotin-avidin system exhibit robust macrophage-modulating and repair-promoting functions and suggest a universal approach for the development of MSC-EX-functionalized biomaterials for advanced therapies.

Periodontitis exacerbates atherosclerosis through Fusobacterium nucleatum-promoted hepatic glycolysis and lipogenesis.

AIMS: Positive associations between periodontitis (PD) and atherosclerosis have been established, but the causality and mechanisms are not clear. We aimed to explore the causal roles of PD in atherosclerosis and dissect the underlying mechanisms. METHODS AND RESULTS: A mouse model of PD was established by ligation of molars in combination with application of subgingival plaques collected from PD patients and then combined with atherosclerosis model induced by treating atheroprone mice with a high-cholesterol diet (HCD). PD significantly aggravated atherosclerosis in HCD-fed atheroprone mice, including increased en face plaque areas in whole aortas and lesion size at aortic roots. PD also increased circulating levels of triglycerides and cholesterol, hepatic levels of cholesterol, and hepatic expression of rate-limiting enzymes for lipogenesis. Using 16S ribosomal RNA (rRNA) gene sequencing, Fusobacterium nucleatum was identified as the most enriched PD-associated pathobiont that is present in both the oral cavity and livers. Co-culture experiments demonstrated that F. nucleatum directly stimulated lipid biosynthesis in primary mouse hepatocytes. Moreover, oral inoculation of F. nucleatum markedly elevated plasma levels of triglycerides and cholesterol and promoted atherogenesis in HCD-fed ApoE-/- mice. Results of RNA-seq and Seahorse assay indicated that F. nucleatum activated glycolysis, inhibition of which by 2-deoxyglucose in turn suppressed F. nucleatum-induced lipogenesis in hepatocytes. Finally, interrogation of the molecular mechanisms revealed that F. nucleatum-induced glycolysis and lipogenesis by activating PI3K/Akt/mTOR signalling pathway in hepatocytes. CONCLUSIONS: PD exacerbates atherosclerosis and impairs lipid metabolism in mice, which may be mediated by F. nucleatum-promoted glycolysis and lipogenesis through PI3K/Akt/mTOR signalling in hepatocytes. Treatment of PD and specific targeting of F. nucleatum are promising strategies to improve therapeutic effectiveness of hyperlipidaemia and atherosclerosis.

Long non-coding RNA AC018926.2 regulates palmitic acid exposure-compromised osteogenic potential of periodontal ligament stem cells via the ITGA2/FAK/AKT pathway.

Although obesity has been proposed as a risk factor for periodontitis, the influence of excessive fat accumulation on the development of periodontitis and periodontal recovery from disease remains largely unknown. This study investigated the cellular response of periodontal ligament stem cells (PDLSCs) to elevated levels of a specific fatty acid, namely, palmitic acid (PA). The mechanism by which PA exposure compromises the osteogenic potential of cells was also explored. It was found that exposure of PDLSCs to abundant PA led to decreased cell osteogenic differentiation. Given that long non-coding RNAs (lncRNAs) play a key role in the stem cell response to adverse environmental stimuli, we screened the lncRNAs that were differentially expressed in PDLSCs following PA exposure using lncRNA microarray analysis, and AC018926.2 was identified as the lncRNA that was most sensitive to PA. Next, gain/loss-of-function studies illustrated that AC018926.2 was an important regulator in PA-mediated osteogenic differentiation of PDLSCs. Mechanistically, AC018926.2 upregulated integrin α2 (ITGA2) expression and therefore activated ITGA2/FAK/AKT signalling. Further functional studies revealed that inactivation of ITGA2/FAK/AKT signalling by silencing ITGA2 counteracted the pro-osteogenic effect induced by AC018926.2 overexpression. Moreover, the results of bioinformatics analysis and RNA immunoprecipitation assay suggested that AC018926.2 might transcriptionally regulate ITGA2 expression by binding to PARP1 protein. Our data suggest that AC018926.2 may serve as a therapeutic target for the management of periodontitis in obese patients.

The impact of Anatomic Features of Asymptomatic Third Molars on the Pathologies of Adjacent Second Molars: A Cross-sectional Analysis.

BACKGROUND: We aimed to comprehensively examine how the anatomic characteristics of asymptomatic third molars (As-M3s) affect distal pathologies of adjacent second molars (Ad-M2s). MATERIALS AND METHODS: Patients with at least 1 quadrant having intact As-M3s and first and second molars were enrolled. Distal pathologies of Ad-M2s, including caries, pocket depth of 4 mm or more (PD4+), and alveolar bone loss of 3 mm or more (ABL3+), were analysed based on As-M3 status (absent/impacted/nonimpacted). Especially within nonimpacted M3s (N-M3s), the effects of regions (maxillary vs mandibular) and dental intervals (narrow vs wide) on Ad-M2 pathologies were further compared. RESULTS: A total of 248 patients with their 805 quadrants were finally included in this cross-sectional study. The impacted and nonimpacted As-M3s elevated the risk of any distal pathology (caries, PD4+, or ABL3+) of Ad-M2s vs M3 absence with odds ratios (ORs) of 8.33 and 3.27, respectively. Within N-M3s, mandibular regions increased the odds of PD4+ (OR, 1.96); wide dental intervals increased the odds of ABL3+ (OR, 3.01). However, maxillary regions and narrow dental intervals contributed to more severe bone loss in Ad-M2s with ABL3+. CONCLUSIONS: The presence of As-M3 is a risk factor for Ad-M2 pathologies irrespective of impaction status. Within N-M3s, Ad-M2 pathologies are significantly influenced by anatomic characteristics such as regions and dental intervals.

Roles of extracellular vesicles in periodontal homeostasis and their therapeutic potential.

Periodontal tissue is a highly dynamic and frequently stimulated area where homeostasis is easily destroyed, leading to proinflammatory periodontal diseases. Bacteria-bacteria and cell-bacteria interactions play pivotal roles in periodontal homeostasis and disease progression. Several reviews have comprehensively summarized the roles of bacteria and stem cells in periodontal homeostasis. However, they did not describe the roles of extracellular vesicles (EVs) from bacteria and cells. As communication mediators evolutionarily conserved from bacteria to eukaryotic cells, EVs secreted by bacteria or cells can mediate interactions between bacteria and their hosts, thereby offering great promise for the maintenance of periodontal homeostasis. This review offers an overview of EV biogenesis, the effects of EVs on periodontal homeostasis, and recent advances in EV-based periodontal regenerative strategies. Specifically, we document the pathogenic roles of bacteria-derived EVs (BEVs) in periodontal dyshomeostasis, focusing on plaque biofilm formation, immune evasion, inflammatory pathway activation and tissue destruction. Moreover, we summarize recent advancements in cell-derived EVs (CEVs) in periodontal homeostasis, emphasizing the multifunctional biological effects of CEVs on periodontal tissue regeneration. Finally, we discuss future challenges and practical perspectives for the clinical translation of EV-based therapies for periodontitis.

How the Loss of Second Molars Corresponds with the Presence of Adjacent Third Molars in Chinese Adults: A Retrospective Study.

Third molars (M3s) can increase the pathological risks of neighboring second molars (M2s). However, whether the M3 presence affects M2 loss remains unknown. This retrospective study aimed to reveal the reasons for M2 loss and how M2 loss relates to neighboring M3s. The medical records and radiographic images of patients with removed M2(s) were reviewed to analyze why the teeth were extracted and if those reasons were related to adjacent M3s. Ultimately, 800 patients with 908 removed M2s were included. In the included quadrants, 526 quadrants with M3s were termed the M3 (+) group, and the other 382 quadrants without M3s were termed the M3 (−) group. The average age of patients in the M3 (+) group was 52.4 ± 14.8 years and that of the M3 (−) group was 56.7 ± 14.9 years, and the difference between the two groups was statistically significant (p < 0.001). Of the 908 M2s, 433 (47.7%) were removed due to caries and sequelae and 300 (33.0%) were removed due to periodontal diseases. Meanwhile, 14.4% of the M2s with adjacent M3s were removed due to distal caries and periodontitis, which were closely related to the neighboring M3s; this percentage was much lower when M3 were absent (1.8%). Additionally, 42.2% of M3s were removed simultaneously with neighboring M2s. The presence of M3s, regardless of impaction status, was associated with an earlier loss of their neighboring M2s.

Gold nanoparticles targeting the autophagy-lysosome system to combat the inflammation-compromised osteogenic potential of periodontal ligament stem cells: From mechanism to therapy.

Although substantial data indicate that the osteogenic potential of periodontal ligament stem cells (PDLSCs) is compromised under inflammatory conditions, the underlying mechanism remains largely unexplored. In this study, we found that both the autophagy levels and autophagic flux levels were decreased in PDLSCs incubated under inflammatory conditions (I-PDLSCs). Based on the increased expression of LC3 II (at an autophagy level) and decreased accumulation of LC3 II (at an autophagic flux level) in I-PDLSCs, we speculated that the disruption of I-PDLSC autophagy arose from dysfunction of the cellular autophagy-lysosome system. Subsequently, our hypothesis was demonstrated by inhibited autophagosome-lysosome fusion, damaged lysosomal function, and suppressed activation of transcription factor EB (TFEB, a master regulator of the autophagy-lysosome system) in I-PDLSCs and verified by TFEB overexpression in I-PDLSCs. We found that gold nanoparticle (Au NP) treatment rescued the osteogenic potential of I-PDLSCs by restoring the inflammation-compromised autophagy-lysosome system. In this context, Au NP ceased to be effective when TFEB was knocked down in PDLSCs. Our data demonstrate the crucial role of the autophagy-lysosome system in cellular osteogenesis under inflammatory conditions and suggest a new target for rescuing inflammation-induced cell dysfunction using nanomaterials to aid cell biology and tissue regeneration.

Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway.

BACKGROUND: High glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibits abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic differentiation under high glucose and the underlying mechanisms remain unknown. METHODS: Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways. RESULTS: We found that osteogenic differentiation of PDLSCs under high glucose was decreased, and metformin addition enhanced this capacity of differentiation. Furthermore, the results of RNA-seq analysis showed that natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. When the underlying pathways involved were investigated, we found that upregulation of NPR3 can compromise the metformin-enhanced PDLSC osteogenic differentiation and activate the MAPK pathway (especially the p38 MAPK and Erk1/2 pathway), and that inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs under high glucose. CONCLUSIONS: The present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.

Role of molybdenum in material immunomodulation and periodontal wound healing: Targeting immunometabolism and mitochondrial function for macrophage modulation.

Recently, strategies that can target the underlying mechanisms of phenotype change to modulate the macrophage immune response from the standpoint of biological science have attracted increasing attention in the field of biomaterials. In this study, we printed a molybdenum-containing bioactive glass ceramic (Mo-BGC) scaffold as an immunomodulatory material. In a clinically relevant critical-size periodontal defect model, the defect-matched scaffold featured robust immunomodulatory activity, enabling long-term stable macrophage modulation and leading to enhanced regeneration of multiple periodontal tissues in canines. Further studies demonstrated that the regeneration-enhancing function of Mo-BGC scaffold was macrophage-dependent by using canines with host macrophage depletion. To investigate the role of Mo in material immunomodulation, in vitro investigations were performed and revealed that Mo-BGC powder extract, similar to MoO42--containing medium, induced M2 polarization by enhancing the mitochondrial function of macrophages and promoted a cell metabolic shift from glycolysis toward mitochondrial oxidative phosphorylation. Our findings demonstrate for the first time an immunomodulatory role of a Mo-containing material in the dynamic cascade of wound healing. By targeting the immunometabolism and mitochondrial function of macrophages, Mo-mediated immunomodulation provides new avenues for future material design in the field of tissue engineering and regenerative medicine.

LncRNA GACAT2 binds with protein PKM1/2 to regulate cell mitochondrial function and cementogenesis in an inflammatory environment.

Periodontal ligament stem cells (PDLSCs) are a key cell type for restoring/regenerating lost/damaged periodontal tissues, including alveolar bone, periodontal ligament and root cementum, the latter of which is important for regaining tooth function. However, PDLSCs residing in an inflammatory environment generally exhibit compromised functions, as demonstrated by an impaired ability to differentiate into cementoblasts, which are responsible for regrowing the cementum. This study investigated the role of mitochondrial function and downstream long noncoding RNAs (lncRNAs) in regulating inflammation-induced changes in the cementogenesis of PDLSCs. We found that the inflammatory cytokine-induced impairment of the cementogenesis of PDLSCs was closely correlated with their mitochondrial function, and lncRNA microarray analysis and gain/loss-of-function studies identified GACAT2 as a regulator of the cellular events involved in inflammation-mediated mitochondrial function and cementogenesis. Subsequently, a comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) and parallel reaction monitoring (PRM) assays revealed that GACAT2 could directly bind to pyruvate kinase M1/2 (PKM1/2), a protein correlated with mitochondrial function. Further functional studies demonstrated that GACAT2 overexpression increased the cellular protein expression of PKM1/2, the PKM2 tetramer and phosphorylated PKM2, which led to enhanced pyruvate kinase (PK) activity and increased translocation of PKM2 into mitochondria. We then found that GACAT2 overexpression could reverse the damage to mitochondrial function and cementoblastic differentiation of PDLSCs induced by inflammation and that this effect could be abolished by PKM1/2 knockdown. Our data indicated that by binding to PKM1/2 proteins, the lncRNA GACAT2 plays a critical role in regulating mitochondrial function and cementogenesis in an inflammatory environment.

Relationship between Presence of Third Molars and Prevalence of Periodontal Pathology of Adjacent Second Molars: a Systematic Review and Meta-analysis.

OBJECTIVE: To estimate the mean prevalence of periodontal pathology of adjacent second molars (A-M2s) to third molars (M3s) and identify related confounding factors. METHODS: Studies published before August 2020 were systematically searched in the Cochrane Library, EMBASE and MEDLINE databases. We included cross-sectional studies that evaluated the periodontal pathology of A-M2s based on clinical or radiographic examinations at the molar level. Studies employing similar periodontal parameters were pooled. Clinical attachment loss ≥ 3 mm, alveolar bone loss ≥ 3 mm or ≥ 20% root length were defined as early periodontal defects, and at least one site with probing depth ≥ 5 mm was considered as deep periodontal pockets around A-M2s in the data synthesis. RESULTS: Nine studies (14,749 M3s) were ultimately included in the meta-analysis. On average, 19% of A-M2s showed distal early periodontal defects with the presence of M3s (95% confidence interval [95% CI] 9%-35%). Subgroup analyses suggested the prevalence was 32% (95% CI 16%-54%) in the mandible, and the prevalence was higher with nonimpacted M3s (25%, 95% CI 12%-47%) than with impacted M3s (19%, 95% CI 10%-35%). Additionally, the pooled prevalence for deep periodontal pockets around A-M2s was 52% (95% CI 39%-64%). Subgroup analyses suggested the prevalence was higher in the mandible (62%, 95% CI 45%-76%) than in the maxilla (43%, 95% CI 31%-56%), and for nonimpacted M3s the prevalence reached 50% (95% CI 36%-64%). CONCLUSION: The presence of M3s, especially mandibular and nonimpacted M3s, negatively affects the periodontal status of A-M2s.

Research progress on the hedgehog signalling pathway in regulating bone formation and homeostasis.

Bone formation is a complex regeneration process that was regulated by many signalling pathways, such as Wnt, Notch, BMP and Hedgehog (Hh). All of these signalling have been demonstrated to participate in the bone repair process. In particular, one promising signalling pathway involved in bone formation and homeostasis is the Hh pathway. According to present knowledge, Hh signalling plays a vital role in the development of various tissues and organs in the embryo. In adults, the dysregulation of Hh signalling has been verified to be involved in bone-related diseases in terms of osteoarthritis, osteoporosis and bone fracture; and during the repair processes, Hh signalling could be reactivated and further modulate bone formation. In this chapter, we summarize our current understanding on the function of Hh signalling in bone formation and homeostasis. Additionally, the current therapeutic strategies targeting this cascade to coordinate and mediate the osteogenesis process have been reviewed.