Carbon Nanotubes Induce Mineralization of Human Cementoblasts.

INTRODUCTION: Carbon nanotubes (CNT) are 1 of the allotropes of carbon with unique properties. CNT shows good bone-tissue compatibility and has been reported to induce osteogenesis; therefore, it is regarded as an ideal material in a wide range of applications. However, the therapeutic effect of CNT-containing materials in the healing of apical periodontal tissue is unknown. The purpose of this study was to clarify the effect of CNT on the proliferation and mineralization of the human cementoblast cell line (HCEM). METHODS: The proliferation of HCEM cells with CNT stimulation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed from 24-72 hours. Calcium deposition levels were evaluated by alizarin red S staining on days 7 and 10, and mineralization-related gene expression was examined by quantitative real-time polymerase chain reaction on days 3, 7, and 10. Scanning electron microscopy was used to observe the culture with CNT on day 14. RESULTS: CNT showed no cytotoxicity to HCEM cell proliferation. Treatment was performed with mineralization medium, CNT-induced HCEM mineralization on day 7, and increased calcium deposition on days 7 and 14. Messenger RNA expression of alkaline phosphatase was significantly increased throughout the experimental period, and bone sialoprotein was significantly increased on day 3 by CNT, whereas no effect was found on mRNA expression of type I collagen. CNT was observed in attachment to the cell surface on day 14. CONCLUSIONS: CNT promotes the mineralization of HCEM cells, indicating the potential as a new bioactive component for apical periodontal tissue regeneration materials through the regulation of cementoblast mineralization.

Periodontal tissue regeneration with cementogenesis after application of brain-derived neurotrophic factor in 3-wall inflamed intra-bony defect.

OBJECTIVE: The purpose of this study is to investigate regenerative process by immunohistochemical analysis and evaluate periodontal tissue regeneration following a topical application of BDNF to inflamed 3-wall intra-bony defects. BACKGROUND: Brain-derived neurotrophic factor (BDNF) plays a role in the survival and differentiation of central and peripheral neurons. BDNF can regulate the functions of non-neural cells, osteoblasts, periodontal ligament cells, endothelial cells, as well as neural cells. Our previous study showed that a topical application of BDNF enhances periodontal tissue regeneration in experimental periodontal defects of dog and that BDNF stimulates the expression of bone (cementum)-related proteins and proliferation of human periodontal ligament cells. METHODS: Six weeks after extraction of mandibular first and third premolars, 3-wall intra-bony defects were created in mandibular second and fourth premolars of beagle dogs. Impression material was placed in all of the artificial defects to induce inflammation. Two weeks after the first operation, BDNF (25 and 50 µg/mL) immersed into atelocollagen sponge was applied to the defects. As a control, only atelocollagen sponge immersed in saline was applied. Two and four weeks after the BDNF application, morphometric analysis was performed. Localizations of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by immunohistochemical analysis. RESULTS: Two weeks after application of BDNF, periodontal tissue was partially regenerated. Immunohistochemical analyses revealed that cells on the denuded root surface were positive with OPN and PCNA. PCNA-positive cells were also detected in the soft connective tissue of regenerating periodontal tissue. Four weeks after application of BDNF, the periodontal defects were regenerated with cementum, periodontal ligament, and alveolar bone. Along the root surface, abundant OPN-positive cells were observed. Morphometric analyses revealed that percentage of new cementum length and percentage of new bone area of experimental groups were higher than control group and dose-dependently increased. CONCLUSION: These findings suggest that BDNF could induce cementum regeneration in early regenerative phase by stimulating proliferation of periodontal ligament cells and differentiation into periodontal tissue cells, resulting in enhancement of periodontal tissue regeneration in inflamed 3-wall intra-bony defects.

How does radiotherapy affect the adhesion of universal adhesive to enamel and dentin? A qualitative and quantitative analysis? / Efecto de la radioterapia a la adhesión del adhesivo universal al esmalte y dentina? Un análisis cualitativo y cuantitativo?

Abstract This study investigated the shear bond strength (SBS) of a universal adhesive to enamel/dentin; 1) that had undergone radiotherapy (RT) and 2) that were readily restored and had undergone RT. Enamel and dentin surfaces were obtained from 90 intact human molars. They were randomly divided into six groups according to the presence and timing of irradiation (RT0:no radiotherapy/control, RT1:RT before restoration, RT2:RT after restoration groups) and adhesive application modes (etch&rinse/ER, self-etch/SE)(n=15). A universal adhesive and resin composites were applied. The radiotherapy protocol was conducted with 60 Gy. The SBS test was subjected (1mm/min) and failure type analysis was performed. The resin-enamel/dentin interfaces were examined. Data were statistically analyzed. For enamel and dentin, the presence and timing of irradiation did not significantly influence the SBS values (p>0.05). For enamel, significantly higher SBS values were obtained using etch&rinse mode than self-etch mode (p<0.05). The predominant failure was mixed type. Loss of enamel prisms and obliterated irregular dentinal tubules were found for the radiotherapy-treated specimens. Resin tags were clearer in the irradiated enamel treated with ER than SE. Irradiation with different timings did not influence the bond strength to enamel and dentin, negatively. Irrespective of radiotherapy, the etch&rinse mode caused higher bond strength to enamel than self-etch mode.

Resumen Este estudio investigó la fuerza de unión al cizallamiento (SBS) de un adhesivo universal al esmalte/dentina en piezas que se habían sometido a radioterapia (RT). Se obtuvieron superficies de esmalte y dentina de 90 molares humanos intactos. Se dividieron aleatoriamente en seis grupos según la presencia y el momento de la irradiación (RT0: sin radioterapia/control, RT1: RT antes de la restauración, RT2: RT después de la restauración considerando los modos de aplicación del adhesivo (n=15). Se aplicó un adhesivo universal y resinas compuestas. El protocolo de radioterapia se realizó con 60 Gy. Se sometió a la prueba adhesiva (1mm/min) y se realizó análisis del tipo de falla. Se examinaron las interfases resina-esmalte/dentina. Los datos fueron analizados estadísticamente. Para el esmalte y la dentina, la presencia y el momento de la irradiación no influyeron significativamente en los valores adhesivos (p>0,05). Para el esmalte, se obtuvieron valores adhesivos significativamente más altos utilizando el modo de grabado y enjuague que el modo de autograbado (p<0,05). La falla predominante fue de tipo mixto. Se encontró pérdida de prismas de esmalte y túbulos dentinarios irregulares obliterados en las muestras tratadas con radioterapia. La irradiación con diferentes tiempos no influyó negativamente en la fuerza de unión al esmalte ni a la dentina. Independientemente de la radioterapia, el modo de grabado ácido y enjuague provocó una mayor fuerza de adhesión al esmalte que el modo de autograbado.

REV-ERBs negatively regulate mineralization of the cementoblasts.

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Intermittent parathyroid hormone promotes cementogenesis via ephrinB2-EPHB4 forward signaling.

Intermittent parathyroid hormone (PTH) promotes periodontal repair, but the underlying mechanisms remained unclear. Recent studies found that ephrinB2-EPHB4 forward signaling mediated the anabolic effect of PTH in bone homeostasis. Considering the similarities between cementum and bone, we aimed to examine the therapeutic effect of PTH on resorbed roots and explore the role of forward signaling in this process. In vivo experiments showed that intermittent PTH significantly accelerated the regeneration of root resorption and promoted expression of EPHB4 and ephrinB2. When the signaling was blocked, the resorption repair was also delayed. In vitro studies showed that intermittent PTH promoted the expression of EPHB4 and ephrinB2 in OCCM-30 cells. The effects of PTH on the mineralization capacity of OCCM-30 cells was mediated through the ephrinB2-EPHB4 forward signaling. These results support the premise that the anabolic effects of intermittent PTH on the regeneration of root resorption is via the ephrinB2-EPHB4 forward signaling pathway.

Oral toxicity to high level sodium fluoride causes impairment of autophagy.

Autophagy is a highly conserved intracellular digestion process that degrades damaged proteins and organelles but the biological roles of autophagy in pathological aspects of oral tissues remain largely unknown. We sought to elucidate the function of autophagy, especially its interplay with apoptosis and oxidative stress, in the oral toxicity induced by exposure to 5 mM sodium fluoride (NaF). Human cementoblasts (HCEM-2) in culture were exposed to 5 mM NaF for 5 min, after which cell viability and cell apoptosis were assessed using the MTS assay and an Annexin V-FITC/PI apoptosis detection kit, respectively. Quantitative RT-PCR and Western blotting were performed to characterize the expression levels of markers for autophagy, apoptosis, and oxidative stress. Senescence-resistant (SAMR1) mice were exposed to 5 mM NaF in their drinking water from 12 to 58 weeks. Micro-computed tomography was used to measure changes in their alveolar bone while immunohistochemistry and immunofluorescence staining was used to evaluate protein expression levels. HCEM-2 cells exposed to 5 mM NaF had decreased levels of autophagy, as shown by reduced expression levels of ATG5, Beclin-1 and LC3-II, elicited apoptosis, which in turn induced oxidative stress and inflammation, as manifested by elevated levels of Bax, cleaved caspase-3, SOD1 and phospho NF-κB. Treatment of mice with 5 mM NaF resulted in histological abnormalities in periodontal tissues, induced excessive oxidative stress and apoptosis, and reduced autophagy. Micro-computed tomography analysis demonstrated that 5 mM NaF caused a decrease in bone areas of mice compared with controls. Exposure to 5 mM NaF induced RANKL (receptor activator of nuclear factor κB ligand) and cathepsin K expression in periodontal tissues, while ATG5 and Beclin-1 expression was abrogated by 5 mM NaF. Taken together, our findings suggest that 5 mM NaF elicits oral toxicity that contributes to excessive apoptosis, oxidative stress, and defective autophagy, which aggravates periodontal tissue damage.

IL1ß inhibits differentiation of cementoblasts via microRNA-325-3p.

Cementum regeneration is considered the gold standard for the treatment of periodontitis. As one of the most important primary proinflammatory cytokines, interleukin 1ß (IL1ß) plays an essential role during the early stage of periodontitis and its amounts simultaneously increase dramatically during this stage. Though promising, the differentiation of cementoblasts upon IL1ß-induced inflammation of the microenvironment and the relative interaction mechanism are still unknown. Here, we found that IL1ß inhibited cementoblast differentiation and microRNA-325-3p (miR-325-3p) was increased during IL1ß-stimulated cementoblasts. Bioinformatics analysis and luciferase reporter assay demonstrated miR-325-3p targeted runt-related transcription factor 2 directly. Transfection of miR-325-3p suppressed cementoblast differentiation in vitro and the formation of cementum-like tissues in vivo. The inhibitor of miR-325-3p could mitigate the above effects induced by IL1ß. Accordingly, our finding suggests a critical role of miR-325-3p in linking inflammation to impaired cementum regeneration and provides a potential possibility for applying miR-325-3p inhibitors in the treatment of periodontitis-related bone loss.

Antagonistic interactions between osterix and pyrophosphate during cementum formation.

During cementum formation, the key roles of osterix (Osx) and inorganic pyrophosphate (PPi), mainly controlled by nucleotide pyrophosphatase 1 (Npp1; encoded by the Enpp1 gene) and progressive ankylosis protein (Ank), have been demonstrated by animal models displaying altered cementum formation. In this study, we analyzed the relationship of Osx and local PPi during cementum formation using compound mutant mice with their wildtype and corresponding single gene mutants. Importantly, functional defects in PPi regulation led to the induction of Osx expression at the cervical cementum as demonstrated by Enpp1 mutant mice and cementoblasts with the retroviral transduction of small hairpin RNA for Enpp1 or Ank. Conversely, cementoblasts exposed to inorganic PPi or with the enforced expression of Enpp1 or Ank reduced Osx expression in a concentration-dependent manner. Furthermore, the loss of Osx induced the higher expression of Npp1 and Ank at the apical region of the developing tooth root as observed in Osx-deficient mice. The activity of PPi-generating ectoenzymes (nucleoside triphosphate pyrophosphohydrolase, NTPPPHase) and the level of extracellular PPi were significantly increased in Osx-knockdown cementoblasts. However, the formation of ectopic cervical cementum was not completely diminished by inactivation of Osx in Enpp1 mutant mice. In addition, fibroblast growth factor (FGF) receptor 1 (Fgfr1) was strongly localized in cementoblasts lining the acellular cementum and involved in the inhibitory regulation of matrix accumulation and further mineralization by supporting PPi production. Taken together, these results suggest that local PPi suppresses matrix accumulation and further mineralization through an antagonistic interaction with Osx under the synergistic influence of FGF signaling during cementum formation.

MicroRNA-181b-5p modulates tumor necrosis factor-α-induced inflammatory responses by targeting interleukin-6 in cementoblasts.

Tooth cementum is a bone-like mineralized tissue and serves as a microbial barrier against invasion and destruction. Cementum is also responsible for tooth stability and defending pulp from outside stimuli, which is formed by cementoblasts. Although it is crucial for periodontal and periapical diseases, the mechanisms underlying the pathophysiological changes of cementoblasts and their inflammatory responses remain unclear. MiR-181b is found to modulate vascular inflammation and endotoxin tolerance. In this study, miR-181b-5p was downregulated in tumor necrosis factor-α (TNF-α)-stimulated cementoblasts, whereas proinflammatory molecules increased. The mouse periapical lesions have similar results, which imitate an inflammatory environment for cementoblasts in vivo. The bioinformatics analysis and dual luciferase reporter assay suggested that miR-181b-5p targeted interleukin-6 (IL-6). Overexpressing miR-181b-5p negatively regulated IL-6 and proinflammatory chemokine. Western blot analysis and luciferase activity reporter assay verified that miR-181b-5p weakened the NF-κB activity. Hence, miR-181b-5p moderated proinflammatory chemokine production by targeting IL-6 in cementoblasts and NF-κB signaling pathway was involved. Furthermore, miR-181b-5p promoted cementoblast apoptosis, which may enhance the resolution of inflammation. Overall, our data revealed that miR-181b-5p was a negative regulator of TNF-α-induced inflammatory responses in cementoblasts.

Lithium chloride attenuates suppressed differentiation induced by mechanical strain in cementoblasts.

Aim: The purpose of this study was to investigate the influence of mechanical strain on OCCM-30 cementoblast differentiation and Wnt/ß-catenin pathway activity. Materials and Methods: Mechanical tension in the form of 2500-µ strain was applied to the cells using the Forcel four-point bending system, with or without the Wnt signaling activator, lithium chloride. Changes in cell differentiation and the expression of Wnt/ß-catenin pathway components in response to strain and lithium chloride were assessed by real-time PCR, immunofluorescence, and western blotting. Results: The mRNA expression levels of the cementoblastogenesis-related genes alkaline phosphatase, runt-related transcription factor 2, and collagen 1, were decreased with mechanical strain. Similarly, the Wnt signaling pathway component genes LRP5, AXIN2, and LEF1 were decreased. The immunofluorescence assay demonstrated that scant ß-catenin underwent nuclear translocation after the cells were subjected to mechanical strain. Moreover, western blotting showed that the protein levels of both ß-catenin and phosphorylated ß-catenin were increased after mechanical strain. In the presence of lithium chloride, the differentiation that was suppressed by mechanical strain was attenuated. Conclusions: 2500-µ strain mechanical strain inhibited cementoblast differentiation activity in vitro, which could be alleviated by actviating Wnt/ß-catenin signaling using lithium chloride.

Periodontal Tissue Regeneration Using Brain-Derived Neurotrophic Factor Delivered by Collagen Sponge.

IMPACT STATEMENT: The various roles played by brain-derived neurotrophic factor (BDNF) in a multitude of tissues and at different scenarios have rendered BDNF a favorable candidate for improving tissue regeneration. Although the tested formulations of BDNF quantitatively regenerate tissue to a level similar to control groups, it resulted in significantly more instances of full regeneration.

Sodium fluoride causes oxidative stress and apoptosis in cementoblasts.

OBJECTIVES: Toothpaste with fluoride concentration up to 5000 ppm are recommended to the patients who are susceptible to root caries; however, the effects of fluoride on cementoblasts have received less attention. METHODS: The OCCM-30 cells were exposed to 0,0.5, 5, 10 mM NaF respectively. A TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay kit was used to detect the DNA fragmentation. Hoechst staining was used to determine changes of nuclear morphology. Real-time quantitative RT-PCR and Western blotting were performed to investigate the mRNA and protein expression of caspase-3,-8,-9, cleaved Poly (ADP-ribose) polymerase (PARP) and Fas-ligand (Fas-L), a ligand of death receptor. CA-DCF-DA [5 (6)-Carboxy-2',7'-dichlorofluorescein diacetate] was used to measure the generation of reactive oxygen species (ROS) in OCCM-30 cells after the NaF stimulation. RESULTS: The results showed apoptotic morphological changes and DNA fragmentation in OCCM-30 cells exposed to high concentration of NaF. 10 mM NaF induced the expression of cleaved caspase-3,-8,-9 and cleaved Poly (ADP-ribose) polymerase (PARP). The mRNA expression of the Fas-L was also increased in cells exposed to 5 mM NaF. Furthermore, 10 mM NaF stimulation resulted in a significant generation of ROS in the OCCM-30 cells. CONCLUSIONS: Our research demonstrated that apoptosis is activated by NaF in OCCM-30 cells through both of the extrinsic death receptor-dependent and oxidative stress-related intrinsic apoptotic pathway. CLINICAL SIGNIFICANCE: More consideration should be given about the fluoride concentration and the frequency of dental products when used to prevent the root caries for patients with gingival recession.

Inhibition of Stat3 signaling pathway decreases TNF-α-induced autophagy in cementoblasts.

Autophagy is a self-digestive process that eliminates impaired or aged proteins and potentially toxic intracellular components to maintain homeostasis. We previously demonstrated that TNF-α played a critical role in cementoblast differentiation, mineralization and apoptosis; however, the effect of TNF-α on cementoblast autophagy has remained unclear. In this study, an elevated immunofluorescence signal of LC3B and autophagic vacuoles, autophagosomes and autolysosomes were detected under TNF-α stimulation in OCCM-30 cells. Autophagy-related genes and proteins, Beclin-1, LC3A and Atg-5, were significantly upregulated by TNF-α in a time- and concentration-dependent manner. During this process, the activity of Stat3 was dramatically enhanced and when the activity of Stat3 was blocked by either a specific chemical inhibitor or siRNA transfection before TNF-α stimulation, the TNF-α-induced upregulation of autophagy-related genes and proteins was strongly inhibited. Our results suggest that TNF-α induced autophagy in cementoblasts was dependent, or partially dependent on the activity of Stat3 signaling pathway.

Remineralization potential of nano-hydroxyapatite on enamel and cementum surrounding margin of computer-aided design and computer-aided manufacturing ceramic restoration.

OBJECTIVE: This study investigates the effects of nano-hydroxyapatite (NHA) gel and Clinpro (CP) on remineralization potential of enamel and cementum at the cavosurface area of computer-aided design and computer-aided manufacturing ceramic restoration. MATERIALS AND METHODS: Thirty extracted human mandibular third molars were sectioned at 1 mm above and below the cemento-enamel junction to remove the cemento-enamel junction portions and replaced them with zirconia ceramic disks by bonding them to the crown and root portions with resin cement. The enamel and cementum with an area of 4×4 mm2 surrounding the ceramic disk was demineralized with carbopol. The demineralized surfaces were treated with either NHA or CP, while 1 group was left with no treatment. Vickers microhardness of enamel and cementum were determined before demineralization, after demineralization, and after remineralization. Analysis of variance and Tukey multiple comparisons were used to determine statistically significant differences at 95% level of confidence. Scanning electron microscopy and X-ray diffraction were used to evaluate for surface alterations. RESULTS: The mean ± SD of Vickers microhardness for before demineralization, after demineralization, and after remineralization for enamel and cementum were 377.37±22.99, 161.95±10.54, 161.70±5.92 and 60.37±3.81, 17.65±0.91, 17.04±1.00 for the no treatment group; 378.20±18.76, 160.72±8.38, 200.08±8.29 and 62.58±3.37, 18.38±1.33, 27.99±2.68 for the NHA groups; and 380.53±25.14, 161.94±5.66, 193.16±7.54 and 62.78±4.75, 19.07±1.30, 24.46±2.02 for the CP groups. Analysis of variance indicated significant increase in microhardness of demineralized enamel and cementum upon the application of either NHA or CP (p<0.05). Post hoc multiple comparisons indicated significantly higher remineralization capability of NHA for both enamel and cementum than CP (p<0.05), as evidenced by scanning electron microscopy, indicating NHA particle deposition in the area of remineralization, and crystallinity accumulation, as indicated by X-ray diffraction. CONCLUSION: NHA gel and CP were capable of remineralization of the enamel and cementum. NHA was more capable in the remineralization process than CP. NHA was extremely capable in the remineralization process for enamel and cementum surrounding the margin of the computer-aided design and computer-aided manufacturing ceramic.

Baicalin Promotes Osteogenic Differentiation of Human Cementoblast Lineage Cells Via the Wnt/ß Catenin Signaling Pathway.

BACKGROUND: Baicalin constitutes a natural bioactive flavonoid extracted from Scutellaria baicalensis Georgi that mediates bone formation. However, the biological functions of baicalin in cementoblasts remain unclear. The purpose of this study was to examine the effects of baicalin on osteogenic differentiation of human cementoblast (HCEM) cells. METHODS: HCEM cells were cultured and treated with 0, 0.01, 0.1 or 1 µM baicalin. Alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) mRNA and protein levels were examined by real-time polymerase chain reaction and western blot analysis, respectively. Cell mineralization was assessed using Alizarin red staining. Glycogen synthase kinase-3 beta (GSK3ß) phosphorylation was measured in 1 µM baicalin-treated HCEM cells with or without the Wnt signaling pathway inhibitor, DKK-1 using ELISA and western blotting. RESULTS: The protein levels of ALP and Runx2 and the intensity of Alizarin red staining were enhanced by baicalin in a dose-dependent manner compared to that of the non-treated control. The ratio of phosphorylated to total GSK3ß increased in the presence of baicalin but was reduced by the addition of DKK-1. Treatment of HCEMs with baicalin up-regulated mRNA levels of ALP and Runx2, which were reduced by DKK-1. In addition, the protein levels of ALP and Runx2, ALP activity, and calcium deposition were also enhanced by baicalin, and these parameters were inhibited by DKK-1. CONCLUSION: Baicalin enhanced osteogenic differentiation of HCEM cells through the Wnt/beta catenin signaling pathway which may be useful for periodontal tissue regeneration.

Overlapping functions of bone sialoprotein and pyrophosphate regulators in directing cementogenesis.

Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PPi), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PPi) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PPi) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp-/-) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp-/- mice and Alpl-/- mice (the latter featuring elevated PPi and OPN), prompted us to examine whether BSP is operating by modulating PPi-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PPi, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PPi in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2µg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PPi by genetic ablation of Ank on the Bsp-/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp-/-; Ank-/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank-/- mice. From these data, we conclude that the perturbations in PPi metabolism are not solely driving the cementum pathology in Bsp-/- mice, and that PPi is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PPi. We propose that BSP and PPi work in concert to direct mineralization in cementum and likely other mineralized tissues.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility.

Intermittent parathyroid hormone (PTH) promotes cementogenesis and alleviates the catabolic effects of mechanical strain in cementoblasts.

BACKGROUND: External root resorption, commonly starting from cementum, is a severe side effect of orthodontic treatment. In this pathological process and repairing course followed, cementoblasts play a significant role. Previous studies implicated that parathyroid hormone (PTH) could act on committed osteoblast precursors to promote differentiation, and inhibit apoptosis. But little was known about the role of PTH in cementoblasts. The purpose of this study was to investigate the effects of intermittent PTH on cementoblasts and its influence after mechanical strain treatment. RESULTS: Higher levels of cementogenesis- and differentiation-related biomarkers (bone sialoprotein (BSP), osteocalcin (OCN), Collagen type I (COL1) and Osterix (Osx)) were shown in 1-3 cycles of intermittent PTH treated groups than the control group. Additionally, intermittent PTH increased alkaline phosphatase (ALP) activity and mineralized nodules formation, as measured by ALP staining, quantitative ALP assay, Alizarin red S staining and quantitative calcium assay. The morphology of OCCM-30 cells changed after mechanical strain exertion. Expression of BSP, ALP, OCN, osteopontin (OPN) and Osx was restrained after 18 h mechanical strain. Furthermore, intermittent PTH significantly increased the expression of cementogenesis- and differentiation-related biomarkers in mechanical strain treated OCCM-30 cells. CONCLUSIONS: Taken together, these data suggested that intermittent PTH promoted cementum formation through activating cementogenesis- and differentiation-related biomarkers, and attenuated the catabolic effects of mechanical strain in immortalized cementoblasts OCCM-30.

Intravenous bisphosphonate therapy does not thicken cementum or change periodontal ligaments of cancer patients.

OBJECTIVE: To test the hypothesis that intravenous (IV) bisphosphonate (BP) therapy thickens or alters the micromorphology of cementum and periodontal ligament (PDL) in cancer patients. STUDY DESIGN: Thirty-two teeth extracted from 24 cancer patients and separated into test (patients who have undergone IV BP therapy, n = 16) and control (patients naive to BP therapy, n = 16) groups were studied. Cementum thickness was measured in 3 different areas of the dental root with polarized light microscopy. PDL was assessed by optical light microscopy and the immunohistochemical expression of periostin. RESULTS: No significant difference was detected in cementum thickness (apical, P = .06; medium, P = .16; cervical, P = .18) between groups. The numbers of fibroblasts in PDL (P = .56), incremental lines of cementum (P = .51) and the immunohistochemical patterns of periostin expression in PDL (P = .68) did not differ between groups. CONCLUSION: IV BP therapy does not thicken cementum or change the micromorphology of PDL.