Axin2+ PDL Cells Directly Contribute to New Alveolar Bone Formation in Response to Orthodontic Tension Force

Wnt–β-catenin signaling plays a key role in orthodontic tooth movement (OTM), a common clinical practice for malocclusion correction. However, its targeted periodontal ligament (PDL) progenitor cells remain largely unclear. In this study, we first showed a synchronized increase in Wnt–β-catenin levels and Axin2+ PDL progenitor cell numbers during OTM using immunostaining of β-catenin in wild-type mice and X-gal staining in the Axin2-LacZ knock-in line. Next, we demonstrated time-dependent increases in Axin2+ PDL progenitors and their progeny cell numbers within PDL and alveolar bones during OTM using a one-time tamoxifen-induced Axin2 tracing line ( Axin2CreERT2/+; R26RtdTomato/+). Coimmunostaining images displayed both early and late bone markers (such as RUNX2 and DMP1) in the Axin2Lin PDL cells. Conversely, ablation of Axin2+ PDL cells via one-time tamoxifen-induced diphtheria toxin subunit A (DTA) led to a drastic decrease in osteogenic activity (as reflected by alkaline phosphatase) in PDL and alveolar bone. There was also a decrease in new bone mass and a significant reduction in the mineral apposition rate on both the control side (to a moderate degree) and the OTM side (to a severe degree). Thus, we conclude that the Axin2+ PDL cells (the Wnt-targeted key cells) are highly sensitive to orthodontic tension force and play a critical role in OTM-induced PDL expansion and alveolar bone formation. Future drug development targeting the Axin2+ PDL progenitor cells may accelerate alveolar bone formation during orthodontic treatment.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway

Activin A, a member of transforming growth factor-β superfamily, is involved in the regulation of cellular differentiation and promotes tissue healing. Previously, we reported that expression of activin A was upregulated around the damaged periodontal tissue including periodontal ligament (PDL) tissue and alveolar bone, and activin A promoted PDL-related gene expression of human PDL cells (HPDLCs). However, little is known about the biological function of activin A in alveolar bone. Thus, this study analyzed activin A-induced biological functions in preosteoblasts (Saos2 cells). Activin A promoted osteoblastic differentiation of Saos2 cells. Activin receptor-like kinase (ALK) 1, an activin type I receptor, was more strongly expressed in Saos2 cells than in HPDLCs, and knockdown of ALK1 inhibited activin A-induced osteoblastic differentiation of Saos2 cells. Expression of ALK1 was upregulated in alveolar bone around damaged periodontal tissue when compared with a nondamaged site. Furthermore, activin A promoted phosphorylation of Smad1/5/9 during osteoblastic differentiation of Saos2 cells and knockdown of ALK1 inhibited activin A-induced phosphorylation of Smad1/5/9 in Saos2 cells. Collectively, these findings suggest that activin A promotes osteoblastic differentiation of preosteoblasts through the ALK1-Smad1/5/9 pathway and could be used as a therapeutic product for the healing of alveolar bone as well as PDL tissue.

Mechanosensitive Piezo1 in Periodontal Ligament Cells Promotes Alveolar Bone Remodeling During Orthodontic Tooth Movement

Orthodontic tooth movement (OTM) is a process depending on the remodeling of periodontal tissues surrounding the roots. Orthodontic forces trigger the conversion of mechanical stimuli into intercellular chemical signals within periodontal ligament (PDL) cells, activating alveolar bone remodeling, and thereby, initiating OTM. Recently, the mechanosensitive ion channel Piezo1 has been found to play pivotal roles in the different types of human cells by transforming external physical stimuli into intercellular chemical signals. However, the function of Piezo1 during the mechanotransduction process of PDL cells has rarely been reported. Herein, we established a rat OTM model to study the potential role of Piezo1 during the mechanotransduction process of PDL cells and investigate its effects on the tension side of alveolar bone remodeling. A total of 60 male Sprague-Dawley rats were randomly assigned into three groups: the OTM + inhibitor (INH) group, the OTM group, and the control (CON) group. Nickel-titanium orthodontic springs were applied to trigger tooth movement. Mice were sacrificed on days 0, 3, 7, and 14 after orthodontic movement for the radiographic, histological, immunohistochemical, and molecular biological analyses. Our results revealed that the Piezo1 channel was activated by orthodontic force and mainly expressed in the PDL cells during the whole tooth movement period. The activation of the Piezo1 channel was essential for maintaining the rate of orthodontic tooth movement and facilitation of new alveolar bone formation on the tension side. Reduced osteogenesis-associated transcription factors such as Runt-related transcription factor 2 (RUNX2), Osterix (OSX), and receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio were examined when the function of Piezo1 was inhibited. In summary, Piezo1 plays a critical role in mediating both the osteogenesis and osteoclastic activities on the tension side during OTM.

Induction of Apoptotic Cell Death by Oral Streptococci in Human Periodontal Ligament Cells

Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells. Streptococcus gordonii showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since S. gordonii increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as Streptococcus mitis, Streptococcus sanguinis, and Streptococcus sobrinus also induced apoptosis, whereas Streptococcus mutans did not. All streptococci tested except S. mutans triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by S. gordonii was not recovered by the ROS inhibitor, resveratrol or n-acetylcysteine. Instead, hydrogen peroxide (H2O2) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except S. mutans generated H2O2, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient S. gordonii mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from S. gordonii alone were sufficient to induce cytotoxicity. Notably, S. gordonii lipoproteins did not induce H2O2 or ROS but cooperatively induced cell death when co-treated with H2O2. Taken together, these results suggest that most oral streptococci except S. mutans efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H2O2 and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.

Network Meta-Analysis of 10 Storage Mediums for Preserving Avulsed Teeth

Many storage mediums are available for the storage of avulsed teeth to preserve the viability of periodontal ligament (PDL) cells before replantation; however, it is unclear which medium is the optimal option. We performed this network meta-analysis to answer this question. A comprehensive search was conducted in PubMed, EMBASE, and the Cochrane library to capture eligible studies investigating the comparative efficacy of Hank's balanced salt solution (HBSS), aloe vera gel (AVG), oral rehydration solution (ORS), coconut water, egg white, green tea, propolis, saline, milk, and water. Statistical analysis was conducted using Review Manager v5.3 and ADDIS v1.16.8. In total, 20 RCTs involving 31 reports were included finally. Direct meta-analysis suggested that HBSS was superior to ORS, milk, saline, and water, ORS was superior to milk but inferior to coconut water and propolis, egg white was superior to milk but inferior to AVG and propolis, propolis was superior to AVG, milk, and saline, and coconut water and water was inferior to saline and milk, respectively. Network meta-analysis suggested that AVG was inferior to the other nine mediums, and propolis was superior to HBSS (SMD, −5260.24; 95% CrI, −10447.39 to −70.37) and milk (SMD, −5461.11; 95% CrI, −10574.99 to −328.51). Moreover, ranking probabilities indicated the highest probability for propolis, followed by saline, ORS, HBSS, milk, egg white, water, green tea, and AVG successively. Propolis may be the optimal media for storing avulsed teeth before replantation. However, given the availability of propolis and HBSS and the hypotonic properties of saline, ORS or milk should also be preferentially selected.

Although Anatomically Micrometers Apart: Human Periodontal Ligament Cells Are Slightly More Active in Bone Remodeling Than Alveolar Bone Derived Cells

The periodontal ligament (PDL) and the alveolar bone are part of the periodontium, a complex structure that supports the teeth. The alveolar bone is continuously remodeled and is greatly affected by several complex oral events, like tooth extraction, orthodontic movement, and periodontitis. Until now, the role of PDL cells in terms of osteogenesis and osteoclastogenesis has been widely studied, whereas surprisingly little is known about the bone remodeling capacity of alveolar bone. Therefore, the purpose of this study was to compare the biological character of human alveolar bone cells and PDL cells in terms of osteogenesis and osteoclastogenesis in vitro. Paired samples of PDL cells and alveolar bone cells from seven patients with compromised general and oral health were collected and cultured. Bone A (early outgrowth) and bone B (late outgrowth) were included. PDL, bone A, bone B cell cultures all had a fibroblast appearance with similar expression pattern of six mesenchymal markers. These cultures were subjected to osteogenesis and osteoclastogenesis assays. For osteoclastogenesis assays, the cells were co-cultured with peripheral blood mononuclear cells, a source for osteoclast precursor cells. The total duration of the experiments was 21 days. Osteogenesis was slightly favored for PDL compared to bone A and B as shown by stronger Alizarin red staining and higher expression of RUNX2 and Collagen I at day 7 and for ALP at day 21. PDL induced approximately two times more osteoclasts than alveolar bone cells. In line with these findings was the higher expression of cell fusion marker DC-STAMP in PDL-PBMC co-cultures compared to bone B at day 21. In conclusion, alveolar bone contains remodeling activity, but to a different extent compared to PDL cells. We showed that human alveolar bone cells can be used as an in vitro model to study bone remodeling.

STORAGE MEDIUM FOR AVULSED TEETH: A LITERATURE REVIEW

Avulsion injury is one of the most severe types of traumatic dental injuries. Following avulsion, periodontal ligament tissues are injured and the vessels and nerves of the pulp rupture at the apical foramen which causes pulp necrosis. In studies it was reported that the key to retention of the knocked-out teeth was to maintain the viability of the periodontal ligament. Storage media plays an important role in preserving the viability of PDL cells during extra alveolar time. This article highlights the different storage medias available for avulsed teeth, along with their merits and demerits.

Spatial Controls of Ligamentous Tissue Orientations Using the Additively Manufactured Platforms in an In Vivo Model: A Pilot Study

The periodontal ligaments (PDLs) with specific orientations to tooth-root surfaces play a key role in generating biomechanical responses between the alveolar bone and cementum as a tooth-supporting tissue. However, control of angulations and regeneration of the ligamentous tissues within micron-scaled interfaces remains challenging. To overcome this limitation, this study investigated surface fabrications with microgroove patterns to control orientations of rat PDL cells in vitro and fibrous tissues in vivo. After being harvested, rat PDL cells were cultured and three different microgroove patterns (∠PDL groove = 0°, ∠PDL groove = 45°, and ∠PDL groove = 90°) were created by the digital slicing step in 3D printing. Cell-seeded scaffolds were subcutaneously transplanted at 3 and 6 weeks. In histology images, rat PDL cells were spatially controlled to angularly organize following the microgroove patterns and fibrous tissues were formed in scaffolds with specific angulations, which were reflected by additively manufactured microgroove topographies. Based on the results, specifically characterized surface topographies were significant to directly/indirectly organizing rat PDL cell alignments and fibrous tissue orientations. Therefore, interactions between surface topographies and tissue organizations could be one of the key moderators for the multiple tissue complex (bone-ligament-cementum) neogenesis in periodontal tissue engineering.

The Role of RANKL and Involvement of Cementum in Orthodontic Root Resorption

Orthodontic root resorption (ORR) is an unintended side effect of orthodontic treatment, and severe ORR can affect treatment outcome. Receptor activator of nuclear factor kappa-B ligand (RANKL) has been detected in the resorbed cementum and periodontal (PDL) tissues exposed to excessive orthodontic forces. Recent studies have demonstrated that PDL cells and cementoblasts express RANKL that may play a role in ORR during orthodontic tooth movement. It is known that the hardness of cementum in human maxillary premolars differs among individuals. Furthermore, this difference has been reported to be attributed to differences in the calcium (Ca)/phosphorus (P) ratio. A correlation was observed between the Vickers hardness and Ca/P ratio of the cementum in the apical region. These findings suggest that cementum hardness and the Ca/P ratio may be indirectly involved in ORR caused by orthodontic forces. In this review, it aims to identify the role of RANKL and involvement of cementum in ORR.