Hypoxic dental pulp stem cells-released succinate promotes osteoclastogenesis and root resorption.

Introduction: Clinical studies have shown that endodontically-treated nonvital teeth exhibit less root resorption during orthodontic tooth movement. The purpose of this study was to explore whether hypoxic dental pulp stem cells (DPSCs) can promote osteoclastogenesis in orthodontically induced inflammatory root resorption (OIIRR). Methods: Succinate in the supernatant of DPSCs under normal and hypoxic conditions was measured by a succinic acid assay kit. The culture supernatant of hypoxia-treated DPSCs was used as conditioned medium (Hypo-CM). Bone marrow-derived macrophages (BMDMs) from succinate receptor 1 (SUCNR1)-knockout or wild-type mice were cultured with conditioned medium (CM), exogenous succinate or a specific inhibitor of SUCNR1 (4c). Tartrate-resistant acid phosphatase (TRAP) staining, Transwell assays, qPCR, Western blotting, and resorption assays were used to evaluate osteoclastogenesis-related changes. Results: The concentration of succinate reached a maximal concentration at 6 h in the supernatant of hypoxia-treated DPSCs. Hypo-CM-treated macrophages were polarized to M1 proinflammatory macrophages. Hypo-CM treatment significantly increased the formation and differentiation of osteoclasts and increased the expression of osteoclastogenesis-related genes, and this effect was inhibited by the specific succinate inhibitor 4c. Succinate promoted chemotaxis and polarization of M1-type macrophages with increased expression of osteoclast generation-related genes. SUCNR1 knockout decreased macrophage migration, M1 macrophage polarization, differentiation and maturation of osteoclasts, as shown by TRAP and NFATc1 expression and cementum resorption. Conclusions: Hypoxic DPSC-derived succinate may promote osteoclast differentiation and root resorption. The regulation of the succinate-SUCNR1 axis may contribute to the reduction in the OIIRR.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

NLRP3-mediated periodontal ligament cell pyroptosis promotes root resorption.

AIM: To investigate the mechanisms by which periodontal ligament cells (PDLCs) convert biomechanical stimulation into inflammatory microenvironment inducing root resorption (RR). MATERIALS AND METHODS: RNA sequencing was employed to explore mechanisms in force-inflammatory signal transduction. Then resorption volume, odontoclastic activity, PDLC pyroptotic ratio and NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis pathway activation were analysed under force and pyroptosis inhibition. Further osteoclast formation, macrophage number and transwell polarization demonstrated the effects of PDLC pyroptosis on osteoclastogenesis and M1 polarization. RESULTS: RNA sequencing revealed that NLRP3-mediated PDLC pyroptosis induced by Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NFκB)/NLRP3 pathway may be involved in mechano-inflammatory signal transduction. PDLC pyroptosis under force and the expression of NLRP3-mediated pyroptosis pathway in force-enhanced PDLCs were significantly increased, both in vivo and in vitro. MCC950 administration was sufficient to reduce PDLC pyroptosis and alleviate RR, odontoclast formation and M1 polarization in vivo. Further in vitro exploration showed that MCC950 treatment reduced PDLC force-promoted pyroptosis and blocked NLRP3-mediated pyroptosis pathway. Moreover, by treating THP-1 with force-pretreated PDLCs or supernatants, NLRP3-mediated PDLC pyroptotic released products induced osteoclast formation and M1 polarization. CONCLUSIONS: NLRP3-mediated PDLC pyroptosis promotes RR. PDLCs transmit excessive force into inflammation signals through TLR4/NFκB/NLRP3 pathway, inducing PDLC pyroptosis, which directly promotes odontoclast formation and subsequent RR or promotes M1 polarization to indirectly trigger odontoclastogenesis and RR.

Stem cells derived from human exfoliated deciduous teeth-based media in a rat root resorption model.

OBJECTIVE: Root resorption may occur during orthodontic treatment. Herein, we investigated the effect of a culture supernatant of stem cells derived from human exfoliated deciduous teeth on root resorption. DESIGN: Twelve 8-week-old male Sprague-Dawley rats were used, and their maxillary first molars were pulled with excessive orthodontic force to induce root resorption. On days 1 and 7 after traction initiation, stem cells derived from human exfoliated deciduous teeth and alpha minimum essential medium (control group) were administered. After 14 days, the maxillary bone was evaluated for tooth movement. The expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6, and interleukin 17 was evaluated on the compression side and tension side. RESULTS: No significant difference in tooth movement was observed between the two groups. Root resorption decreased in the group administered the culture supernatant compared with in the control. Immunohistochemical staining revealed increased osteoprotegerin expression and decreased receptor activators for nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6, and interleukin 17 on the compression side and tension side. CONCLUSIONS: Administration of stem cells derived from human exfoliated deciduous teeth affected the expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6 and interleukin 17; hence, these stem cells may inhibit root resorption by regulating their expression.

Force-Loaded Cementocytes Regulate Osteoclastogenesis via S1P/S1PR1/Rac1 Axis.

Orthodontically induced inflammatory root resorption (OIIRR) is the major iatrogenic complication of orthodontic treatment, seriously endangering tooth longevity and impairing masticatory function. Osteoclasts are thought to be the primary effector cells that initiate the pathological process of OIIRR; however, the cellular and molecular mechanisms responsible for OIIRR remain unclear. Our previous studies revealed that cementocytes, the major mechanically responsive cells in cementum, respond to compressive stress to activate and influence osteoclasts locally. For this study, we hypothesized that the sphingosine-1-phosphate (S1P) signaling pathway, a key mechanotransduction pathway in cementocytes, may regulate osteoclasts under the different magnitudes of either physiologic compressive stress that causes tooth movement or pathologic stress that causes OIIRR. Here, we show a biphasic effect of higher compression force stimulating the synthesis and secretion of S1P, whereas lower compression force reduced signaling in IDG-CM6 cementocytes. Using conditioned media from force-loaded cementocytes, we verified the cell-to-cell communication between cementocytes and osteoclasts and show that selective knockdown of S1PR1 and Rac1 plays a role in cementocyte-driven osteoclastogenesis via the S1P/S1PR1/Rac1 axis. Most importantly, the use of inhibitors of this axis reduced or prevented the pathological process of OIIRR. The intercellular communication mechanisms between cementocytes and osteoclasts may serve as a promising therapeutic target for OIIRR.

Programmed cell death of periodontal ligament cells.

The periodontal ligament is a crucial tissue that provides support to the periodontium. Situated between the alveolar bone and the tooth root, it consists primarily of fibroblasts, cementoblasts, osteoblasts, osteoclasts, periodontal ligament stem cells (PDLSCs), and epithelial cell rests of Malassez. Fibroblasts, cementoblasts, osteoblasts, and osteoclasts are functionally differentiated cells, whereas PDLSCs are undifferentiated mesenchymal stem cells. The dynamic development of these cells is intricately linked to periodontal changes and homeostasis. Notably, the regulation of programmed cell death facilitates the clearance of necrotic tissue and plays a pivotal role in immune response. However, it also potentially contributes to the loss of periodontal supporting tissues and root resorption. These findings have significant implications for understanding the occurrence and progression of periodontitis, as well as the mechanisms underlying orthodontic root resorption. Further, the regulation of periodontal ligament cell (PDLC) death is influenced by both systemic and local factors. This comprehensive review focuses on recent studies reporting the mechanisms of PDLC death and related factors.

Periodontal Fibroblasts-Macrophage Crosstalk in External Inflammatory Root Resorption.

INTRODUCTION: This study aimed to understand the influence of periodontal fibroblasts (PDLFs) on clastic differentiation of macrophages (Mφ) in different resorptive environments. METHODS: PDLF-Mφ direct coculture (juxtacrine) was seeded on dentin, cementum, and polystyrene with/without lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand for 7 and 14 days and stained for tartrate-resistant acid phosphatase (TRAP) activity. PDLF-Mφ cocultured on polystyrene were immunostained for CD80, CD206, NFATc1, STAT6, and periostin, and cell culture supernatants were assessed for cytokines on days 2 and 7. Mφ grown in conditioned media of PDLFs (paracrine) and Mφ monoculture were used as controls. Data was analyzed using Student t test and one-way analysis of variance with the Tukey multiple comparisons test (P < .05). RESULTS: PDLF-Mφ coculture showed a higher number of TRAP-positive multinucleated cells than Mφ monoculture on dentin and polystyrene. No TRAP-positive multinucleated cells were observed in paracrine and cementum. The expression of CD80 and CD206 in PDLF-Mφ was similar at day 2, whereas CD206 was greater than CD80 at day 7. The expression of STAT6 was greater than NFATc1 at both days 2 and 7 (P < .05). Periostin expression in the presence of the lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand combination was down-regulated in PDLF monoculture, whereas it was up-regulated in PDLF-Mφ coculture. The cytokine profile of PDLF-Mφ on day 2 was predominated by interleukin (IL)-1ß, tumor necrosis factor alpha, and MMP9 and MMP2 on day 7. IL-6 and IL-8 showed steady expression at both days 2 and 7. CONCLUSIONS: The study highlights the juxtacrine effect of PDLFs on the clastic differentiation of Mφ with a difference in clastic activity between dentin and cementum. The study also emphasizes the temporal effect of tumor necrosis factor alpha, MMP2, MMP9, and IL-1ß on intercellular crosstalk in resorptive environments.

Difference in apical resorption activity during rat molar root formation in response to mechanical force.

OBJECTIVE: To investigate whether there is a difference in apical resorption activity during the development of roots in response to mechanical force in vivo. METHODS: Maxillary first molars (M1) from postnatal day (PN) 21 and PN35 male rats were selected as representatives of the root-developing and root-completing groups, respectively. A mechanical force of 3 cN was applied to M1 on PN21 and PN35, and the maxilla was collected on PN28 and PN42. Odontoclastogenesis and root morphology were investigated using micro-focus X-ray computed tomography, followed by immunohistochemistry and quantitative real-time polymerase chain reaction to clarify root resorption activity. RESULTS: Development of the mesiobuccal root (MBR) preceded the mesial root (MR). In the PN28 force application (FA) group, the dentine was bent, but the histology, including Hertwig's epithelial root sheath (HERS), was intact. No odontoclasts and resorption lacunae were found in the apical area of the MRs, and only lateral root resorption was observed. External apical root resorption (EARR) was observed in the MR of PN42 (FA) and in the MBR of both PN28 (FA) and PN42 (FA). The expression of osteopontin changed accordingly. No significant change occurred in osteoprotegerin or receptor activator of nuclear factor-κB ligand expression in the MRs of the PN28 (FA) group. LIMITATIONS: Our animal model did not adequately simulate the clinical process of tooth movement in humans. CONCLUSIONS: Force application delayed HERS dissociation on the compression side of the developing roots, leading to inhibitory effects on cementogenesis, which resulted in decreased odontoclast differentiation and prevention of EARR.

Dynamic alternations of RANKL/OPG ratio expressed by cementocytes in response to orthodontic­induced external apical root resorption in a rat model.

The aim of the present study was to investigate the alterations in the formation of cementocytes in response to orthodontic forces and to evaluate the contribution of these cells in the biological changes of tooth movement and associated root resorption. A total of 90 Sprague Dawley rats were randomly assigned to the control, high force, and low force groups. Intrusion forces of 10 and 50 g were applied on the rat molar to induce tooth intrusion. The tooth movement was observed from 0 to 14 days by micro­computed tomography, bone histometric analysis, tartrate­resistant acid phosphatase staining, as well as reverse transcription­quantitative PCR and immunofluorescence staining assays. The results suggested that under low force conditions, osteoclasts were distributed at a higher frequency on the bone side than on the root side. Under high force conditions, both sides suffered osteoclast infiltration. In the low force group, the cementocytes exhibited downregulated sclerostin (SOST) and osteoprotegerin (OPG) mRNA levels and a lower receptor activator of nuclear factor­κB ligand (RANKL)/OPG ratio over a certain period of time. The expression levels of these genes were lower compared with those of the osteocytes at each time­point. In the high force group, both cementocytes and osteocytes upregulated the SOST and RANKL/OPG ratio on days 7 and 14, while the cementocytes expressed higher levels of SOST mRNA than those noted in the osteocytes. These data suggested that cementocytes responded to the orthodontic force via modulation of the RANKL/OPG ratio and SOST expression. The biological response of cementocytes contributed to the mechanotransduction and homoeostasis of the roots under compression. Excessive forces may act as a negative factor of this regulatory role. These results expand our knowledge on the function of cementocytes.

Lithium reduces orthodontically induced root resorption by suppressing cell death, hyalinization, and odontoclast formation in rats.

OBJECTIVES: To examine whether lithium suppresses orthodontically induced root resorption (OIRR) via two mechanisms (prevention of hyalinization in periodontal tissue and suppression of odontoclasts) and to investigate the changes in the periodontal tissue and alveolar bone, focusing on the appearance of cell death, hyalinization, and odontoclasts. MATERIALS AND METHODS: The maxillary first molars of 10-week-old male Wistar rats were moved mesially by a closed-coil spring for 14 days. Lithium chloride (LiCl; 0.64 mM/kg) or saline (control) was administered intraperitoneally daily. Tooth movements were measured using micro-computed tomography. Appearances of cell death, hyalinization, and odontoclasts were evaluated by histological analysis. RESULTS: OIRR observed on day 14 in the control group was suppressed strongly by LiCl administration. Apoptotic cells observed on day 1 in the compression area were gradually diminished on days 2 and 3 and transformed to hyalinization tissue in the control group. LiCl administration remarkably suppressed this cell death and subsequent hyalinization. Also, the appearance of odontoclasts in the compression area observed on day 7 was significantly suppressed by LiCl administration. Accordingly, these degenerative processes to OIRR were suppressed substantially by LiCl treatment. CONCLUSIONS: Lithium reduces OIRR through the suppression of periodontal ligament cell death, hyalinization, and odontoclast formation.

CXCL12/CXCR4 Mediates Orthodontic Root Resorption via Regulating the M1/M2 Ratio.

Mechanical force-induced external root resorption is a major clinical side effect of orthodontic treatment. Recent work has revealed that M1 macrophages play a vital role in promoting orthodontic root resorption (ORR), but the mechanism of how mechanical force stimulation increases the M1/M2 macrophage ratio in periodontal tissue is poorly understood. In the current study, we showed that C-X-C motif chemokine 12 (CXCL12)+ periodontal ligament cells (PDLCs) and C-X-C chemokine receptor type 4 (CXCR4)+ monocytes in the periodontal ligament (PDL) were significantly increased after force application with ongoing root resorption, and these effects were partially rescued after force removal in mice. The expression of CXCL12 in PDLCs was increased by force stimulation in a time- and intensity-dependent manner in vitro. Blockage of the CXCL12/CXCR4 axis using CXCR4 antagonist AMD3100 was sufficient to alleviate ORR and reverse the force-enhanced M1/M2 macrophage ratio. Further mechanism exploration showed that Ly6Chi inflammatory monocytes homed in a CXCL12/CXCR4 axis-dependent manner. The number and proportion of CD11b+ Ly6Chi inflammatory monocytes in cervical lymph nodes were significantly increased by force loading, accompanied by decreased CD11b+ Ly6Chi monocytes in the blood. These changes were blunted by intraperitoneal injection of AMD3100. In addition, blockage of the CXCL12/CXCR4 axis effectively reversed M2 suppression and promoted M1 polarization. Collectively, results indicate that force-induced CXCL12/CXCR4 axis mediates ORR by increasing the M1/M2 ratio in periodontal tissues through attracting Ly6Chi inflammatory monocytes and modulating macrophage polarization. The results also imply that AMD3100 is potentially inhibitory to root resorption.

Microcracks on the Rat Root Surface Induced by Orthodontic Force, Crack Extension Simulation, and Proteomics Study.

Root resorption is a common complication during orthodontic treatment. Microcracks occur on the root surface after an orthodontic force is applied and may be related to the root resorption caused by the orthodontic process. However, the mechanisms underlying root resorption induced by microcracks remain unclear. In this study, a rat orthodontic model was used to investigate the biological mechanisms of root resorption caused by microcracks. First, the first molar was loaded with 0.5-N orthodontic force for 7 days, and microcracks were observed on the root apex surface using a scanning electron microscope. Second, to describe the mechanical principle resulting in microcracks, a finite element model of rat orthodontics was established, which showed that a maximum stress on the root apex can cause microcrack extension. Third, after 7 days of loading in vivo, histological observation revealed that root resorption occurred in the stress concentration area and cementoclasts appeared in the resorption cavity. Finally, proteomics analysis of the root apex area, excluding the periodontal ligament, revealed that the NOX2, Aifm1, and MAPK signaling pathways were involved in the root resorption process. Microcrack extension on the root surface increases calcium ion concentrations, alters the proteins related to root resorption, and promotes cementoclast formation.

Effect of a DPP-4 Inhibitor on Orthodontic Tooth Movement and Associated Root Resorption.

OBJECTIVES: Dipeptidyl peptidase-4 (DPP-4) inhibitors are used as a treatment for type 2 diabetes mellitus and have also recently been applied to enhance bone quality and density, and increase the expression of bone markers. This study aimed to investigate the effect of a DPP-4 inhibitor on orthodontic tooth movement (OTM) and related root resorption in a mouse model. MATERIALS AND METHODS: Mice were randomly divided into three groups: those undergoing OTM with the addition of a DPP-4 inhibitor (30 µg), those undergoing OTM and receiving phosphate-buffered saline (PBS), and those without force loading (control group). OTM was achieved by means of a nickel-titanium closed coil spring that moved the first molar in a mesial direction for 12 days. The distance of OTM was measured using silicone impression. Maxillae were removed for histological analysis or real-time PCR analysis. RESULTS: The distance of OTM and the number of osteoclasts were significantly decreased after administration of the DPP-4 inhibitor, which also significantly suppressed the number of odontoclasts and root resorption after OTM. Furthermore, the mRNA expression of tumour necrosis factor-α (TNF-α) and the receptor activator of nuclear factor kappa-B ligand (RANKL) were decreased in DPP-4 inhibitor-treated mice compared with those receiving PBS and control animals. CONCLUSION: The DPP-4 inhibitor inhibited tooth movement and associated root resorption by blocking the formation of osteoclasts and odontoclasts, respectively. It also appeared to inhibit osteoclastogenesis and odontoclastogenesis by suppressing the expression of TNF-α and/or RANKL.

A New Luminescent Zn(II) Complex: Selective Sensing of Cr2O72- and Prevention Activity Against Orthodontic Root Absorption by Suppressing Inflammatory Response.

A novel luminescent coordination polymer (CP) based on Zn(II) ions as nodes [Zn(OPY)1.5(Hbtc)]n (1), [H3btc = trimesic acid and OPY = 4,4'-(oxybis(4,1-phenylene))dipyridine] has been prepared via the solvothermal assembly of a tripodal multicarboxylic acid ligand, a bis-pyridyl ligand with V-shape containing two diverse coordination patterns as well as Zn2 + ion. The experiments of photoluminescence also reflect that the coordination polymer 1 has high sensitivity to potassium dichromate, and its quenching efficiency is Ksv of 2.12 × 104 L·mol- 1. Furthermore, its treatment activity on orthodontic root absorption was evaluated in vivo. Firstly, the CCK-8 assay was performed in this research to evaluate the biotoxicity of the synthetic compound. Next, the TNF-α and Cbfα1 released by the periodontal ligament fibroblast was determined via the ELISA test kit. In addition to this, the signaling pathway of NF-κB activation after treated with compound was measured by the RT-PCR.

Involvement of interleukins-17 and -34 in exacerbated orthodontic root resorption by jiggling force during rat experimental tooth movement.

BACKGROUND: Orthodontically induced root resorption (OIRR) is considered as an undesirable and unpredictable sequel of orthodontic treatment. Recent reports demonstrated that interleukin (IL)-17/IL-34, and T cells secrete inflammatory/osteoclastogenic cytokines, which might stimulate osteoclastogenesis/bone resorption. However, little is known about the role played by IL-17/IL-34 in OIRR. The present study was aimed at investigating the odontoclastic expression pattern of IL-17 and IL-34 in resorbed cementum during different experimental tooth movements in vivo. METHODS: Twenty-four 8-week-old male Wistar rats were divided into four groups: control group, optimal force group (10 g), heavy force group (50 g), and jiggling force group (compression and tension, repetition; 10 g). After 7, 14, and 21 days, the expression levels of IL-17 and IL-34 protein in the resorbed cementum were analyzed using immunohistochemical methods. RESULTS: On day 21, the immunoreactivity for IL-17 and IL-34 in resorbed roots in the jiggling force group was stronger than that in the heavy force and optimal force groups. Moreover, the number of IL-17-positive and IL-34-positive odontoclasts was significantly increased in the jiggling force group compared with those in the other groups on day 21. CONCLUSIONS: These results suggest that jiggling forces might exacerbate OIRR compared with heavy forces, as evidenced by the increased expression of IL-17 and IL-34 in odontoclasts obtained from resorbed roots.

Knockdown of DANCR reduces osteoclastogenesis and root resorption induced by compression force via Jagged1.

LncRNA DANCR has been proven to be involved in osteoblast differentiation. This study aims to investigate the role of DANCR in osteoclast formation and root resorption in periodontal ligament (PDL) cells induced by compression force (CF). Rat orthodontic tooth movement (OTM) model was established. The molecules expressions in the areas of root resorption form OTM model were measured. The number of osteoclasts was measured using Tartrate-resistant acid phosphatase (TRAP) staining. The bone resorption was detected using pit formation assay. We showed that the expression of DANCR and Jagged1 protein was increased in rat OTM model and human periodontal ligament (hPDL) cells treated with CF, and CF increased the production of Jagged1, RANKL, and IL-6 from the hPDL cells. Moreover, DANCR could positively regulate Jagged1 protein expression. Knockdown of DANCR could change the promotion effect of CF on osteoclastogenesis and bone resorption in vitro and in vivo experiments, while overexpression of Jagged1 reversed si-DANCR effect. Taken together, knockdown of DANCR reduced osteoclast formation and root resorption induced by CF via Jagged1.

Effect of tumor necrosis factor α on ability of SHED to promote osteoclastogenesis during physiological root resorption.

Physiological root resorption of deciduous teeth is a normal phenomenon, however, the potential mechanisms underlying this process remain unclear. This study aimed to investigate ability of stem cells from human exfoliated deciduous teeth (SHED) on promoting the osteoclastic differentiation of osteoclast precursors and clarify mechanisms underlying this process in vitro. SHED and dental pulp stem cells (DPSCs) were obtained from deciduous teeth and healthy permanent teeth. An indirect co-culture system of SHED or DPSCs were used. The osteoclast precursor peripheral blood mononuclear cells (PBMCs) were established. Ability of SHED and DPSCs in promoting osteoclastogenesis was determined using triiodothyronine receptor auxiliary protein (TRAP) staining, real-time real-time PCR (RT-PCR) and western blotting. The effect of inflammation on the pro-osteoclastogenesis ability of SHED was determined using enzyme linked immunosorbent assay (ELISA), RT-PCR and western blotting. The function of the nuclear factor-κB (NF-κB) pathway in promoting the osteoclastogenesis ability of SHED was determined using RT-PCR and western blotting. SHED exhibited an increased ability to promote osteoclastic differentiation. Expression of tumor necrosis factor-α (TNF-α) was significantly higher in SHED than in DPSCs. Expression of cathepsin K (CTSK), TRAP, and receptor-activator of nuclear-factor-κ B ligand (RANKL), RANKL/osteoprotegerin (OPG) ratio, and expression of cytoplasmic phosphorylated inhibitor of NF-κB α (p-IκBα) and nuclear p65 were markedly up-regulated in SHED post the TNF-α treatment but decreased following NF-κB inhibition. In conclusion, inflammatory cytokine TNF-α appeared to activate NF-κB pathway to up-regulate expression of NF-κB, enhancing ability of SHED in promoting osteoclastogenesis via regulating RANKL/OPG expression.

Biomarkers of inflammatory external root resorption as a result of traumatic dental injury to permanent teeth in children.

OBJECTIVE: External inflammatory root resorption (EIRR) is a common complication of traumatic dental injury (TDI) that can be detected radiologically. During EIRR, various proteins are released into gingival sulcus fluid (GCF). The aim of the study was to monitor the levels of selected proteins in GCF in children (8-16 years of age) in order to assess their utility in the early diagnosis of EIRR. DESIGN: Twenty five children who experienced TDI to permanent incisors with ended root development were enrolled. GCF was collected from injured and control teeth with paper strips within seven days after TDI and on three visits during six-month follow-up. Concentrations of IL-1α, IL-1ß, IL-6, IL-8, TNFα, RANKL and MMP-9 in GCF were measured using enzyme-linked immunosorbent assays. EIRR was confirmed by radiological imaging techniques. RESULTS: Of all analyzed proteins, only the levels of IL-1α, Il-1ß and TNFα in GCF from the injured teeth with resorption were higher than in GCF from control teeth on the visit during which the EIRR was diagnosed. In univariate logistic regression model, the concentration of IL-1α in GCF was found as the strongest risk factor for the occurrence of EIRR. CONCLUSIONS: The composition of GCF may be indicative of EIRR after TDI. The monitoring of selected biomarkers in GCF may help to detect EIRR at its early stage and might be useful in reducing radiological exposure in children after TDI. IL-1α can be considered as a potential marker of the EIRR in children after TDI to the permanent teeth.

Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats.

Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 µM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1ß, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1ß, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1ß, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement.

Identification of salivary protein biomarkers for orthodontically induced inflammatory root resorption.

PURPOSE: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most prevalent and unavoidable consequence of orthodontic tooth movement. The aim of this study was to discover potential diagnostic protein biomarkers for detection of OIIRR in whole saliva (WS). MATERIAL AND METHODS: Unstimulated WS was collected from 72 subjects: 48 OIIRR patients and 24 untreated, generally healthy, age and gender matched controls. Radiographic assessment of periapical x-rays of four upper incisors taken before and 9 months after bonding was done. High-abundance proteins were depleted followed by two-dimensional-gel-electrophoresis and quantitative mass spectrometry (qMS). Finally, to initially validate qMS results, Western blotting was performed. RESULTS: qMS revealed differentially expressed proteins in the moderate-to-severe OIIRR group, which have never been found in WS before. Additionally, in the moderate-to-severe young OIIRR group, the pathogenetic mechanisms related to actin cytoskeleton regulation and Fc gamma R- mediated phagocytosis were detected, while in adults- to focal adhesion. Preliminary validation by Western blotting of fetuin-A and p21-ARC indicated expression profile trends similar to those identified by qMS. CONCLUSION: The significance of WS novel proteomic methodologies is clearly demonstrated for detecting new OIIRR biomarkers as well as for unveiling possible novel pathogenetic mechanisms in both young and adult patients.