Mineralization Of Periodontal Ligament Cells Research Articles

SLPI in periodontal Ligament is not sleepy during biophysical force-induced tooth movement.

We aimed to identify a key molecule that maintains periodontal tissue homeostasis during biophysical force-induced tooth movement (BTM) by orchestrating alveolar bone (AB) remodelling. Differential display-PCR was performed to identify key molecules for BTM in rats. To investigate the localization and expression of the identified molecules, immunofluorescence, real-time RT-PCR and Western blotting were performed in rats and human periodontal ligament (PDL) cells. Functional test and micro-CT analysis were performed to examine the in vivo effects of the identified molecules on BTM. Secretory leucocyte peptidase inhibitor (SLPI) in the PDL was revealed as a key molecule for BTM-induced AB remodelling. SLPI was enhanced in the PDL under both compression and tension, and downregulated by an adenyl cyclases inhibitor. SLPI induced osteoblastogenic genes including runt-related transcription factor 2 (Runx2) and synergistically augmented tension-induced Runx2 expression. SLPI augmented mineralization in PDL cells. SLPI induced osteoclastogenic genes including receptor activator of nuclear factor kappa-Β ligand (RANKL) and synergistically augmented the compression-induced RANKL and macrophage colony-stimulating factor (MCSF) expression. Finally, the in vivo SLPI application into the AB significantly augmented BTM. SLPI or its inhibitors might serve as a biological target molecule for therapeutic interventions to modulate BTM.

Asporin Reduces Adult Aortic Valve Interstitial Cell Mineralization Induced by Osteogenic Media and Wnt Signaling Manipulation In Vitro.

Worldwide, calcific aortic valve disease is one of the leading causes of morbidity and mortality among patients with cardiac abnormalities. Aortic valve mineralization and calcification are the key events of adult calcific aortic valve disease manifestation and functional insufficiency. Due to heavy mineralization and calcification, adult aortic valvular cusps show disorganized and dispersed stratification concomitant with deposition of calcific nodules with severely compromised adult valve function. Interestingly, shared gene regulatory pathways are identified between bone-forming cells and heart valve cells during development. Asporin, a small leucine-rich proteoglycan (43 kDa), acts to inhibit mineralization in periodontal ligament cells and is also detected in normal murine adult aortic valve leaflets with unknown function. Therefore, to understand the Asporin function in aortic cusp mineralization and calcification, adult avian aortic valvular interstitial cell culture system is established and osteogenesis has been induced in these cells successfully. Upon induction of osteogenesis, reduced expression of Asporin mRNA and increased expression of bone and osteogenesis markers are detected compared to cells maintained without osteogenic induction. Importantly, treatment with human recombinant Asporin protein reduces the mineralization level in osteogenic media-induced aortic valvular interstitial cells with the concomitant decreased level of Wnt/β-catenin signaling. Overall, all these data are highly indicative that Asporin might be a novel biomolecular target to treat patients of calcific aortic valve disease over current cusp replacement surgery.

Effects of paraoxonase 1 on the cytodifferentiation and mineralization of periodontal ligament cells.

Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P=.0190). PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.

Tensile strength suppresses the osteogenesis of periodontal ligament cells in inflammatory microenvironments.

The present study aimed to investigate the role of orthodontic force in osteogenesis differentiation, matrix deposition and mineralization in periodontal ligament cells (PDLCs) cells in inflammatory microenvironments. The mesenchymal origin of PDLCs was confirmed by vimentin and cytokeratin staining. PDLCs were exposed to inflammatory cytokines (5 ng/ml IL-1β and 10 ng/ml TNF-α) and/or tensile strength (0.5 Hz, 12% elongation) for 12, 24 or 48 h. Cell proliferation and tensile strength-induced cytokine expression were assessed by MTT assay and ELISA, respectively. Runt-related transcription factor 2 (RUNX2) and type I collagen (COL-I) expression were analysed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Additionally, alkaline phosphatase activity was measured, and the mineralization profile was evaluated by alizarin red S staining. PDLCs exposed to tensile strength in inflammatory microenvironments exhibited reduced proliferation and mineralization potential. Treatment with the inflammatory cytokines IL-1β and TNF-α increased RUNX2 expression levels; however, decreased COL-I expression levels, indicating that bone formation and matrix deposition involve different mechanisms in PDL tissues. Notably, RUNX2 and COL-I expression levels were decreased in PDLCs exposed to a combination of an inflammatory environment and loading strength. The decreased osteogenic potential in an inflammatory microenvironment under tensile strength suggests that orthodontic force may amplify periodontal destruction in orthodontic patients with periodontitis.

Effects of Plants on Osteogenic Differentiation and Mineralization of Periodontal Ligament Cells: A Systematic Review.

This systematic review aimed to evaluate the effects of plants on osteogenic differentiation and mineralization of human periodontal ligament cells. The included studies were selected using five different electronic databases. The reference list of the included studies was crosschecked, and a partial gray literature search was undertaken using Google Scholar and ProQuest. The methodology of the selected studies was evaluated using GRADE. After a two-step selection process, eight studies were identified. Six different types of plants were reported in the selected studies, which were Morinda citrifolia, Aloe vera, Fructus cnidii, Zanthoxylum schinifolium, Centella asiatica, and Epimedium species. They included five types of isolated plant components: acemannan, osthole, hesperetin, asiaticoside, and icariin. In addition, some active substances of these components were identified as polysaccharides, coumarins, flavonoids, and triterpenes. The studies demonstrated the potential effects of plants on osteogenic differentiation, cell proliferation, mineral deposition, and gene and protein expression. Four studies showed that periodontal ligament cells induce mineral deposition after plant treatment. Although there are few studies on the subject, current evidence suggests that plants are potentially useful for the treatment of periodontal diseases. However, further investigations are required to confirm the promising effect of these plants in regenerative treatments.

Effects of fluoride on proliferation and mineralization in periodontal ligament cells in vitro.

Fluoride, which is often added to toothpaste or mouthwash in order to protect teeth from decay, may be a novel therapeutic approach for acceleration of periodontal regeneration. Therefore, we investigated the effects of fluoride on proliferation and mineralization in human periodontal ligament cells in vitro. The periodontal ligament cells were stimulated with various concentrations of NaF added into osteogenic inductive medium. Immunohistochemistry of cell identification, cell proliferation, alkaline phosphatase (ALP) activity assay, Alizarin red S staining and quantitative real-time-polymerase chain reaction (RT-PCR) were performed. Moderate concentrations of NaF (50-500 μmol/L) had pro-proliferation effects, while 500 μmol/L had the best effects. ALP activity and calcium content were significantly enhanced by 10 μmol/L NaF with osteogenic inductive medium. Quantitative RT-PCR data varied in genes as a result of different NaF concentrations and treatment periods. We conclude that moderate concentrations of NaF can stimulate proliferation and mineralization in periodontal ligament cells. These in vitro findings may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of suitable concentrations of NaF into the medication for periodontitis treatment, i.e., into periodontal packs and tissue patches.

PLAP-1/Asporin Regulates TLR2- and TLR4-induced Inflammatory Responses

Periodontal ligament–associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.

Periodontal healing with a preameloblast-conditioned medium in dogs.

The predictability of conventional periodontal treatments for damaged periodontal tissue is limited, particularly on the regeneration of new cementum. As signaling molecules, a range of growth factors has been used to promote periodontal regeneration on periodontal ligament (PDL) and cementum defects. A preameloblast-conditioned medium (PA-CM) was prepared from cultured murine apical bud cells, which can differentiate into ameloblasts. We examined the effect of PA-CM on PDL cells and cementoblasts invitro and evaluated histologically the effects of PA-CM on the regeneration of experimentally induced periodontal defects invivo. In vitro, the effects of PA-CM on the migration of human PDL cells were examined using a scratch wound healing assay and a transwell assay. The differentiation and mineralization potential of PA-CM-treated human PDL cells and murine cementoblastic OCCM-30 cells was examined by real-time polymerase chain reaction and Alizarin red-S staining. In vivo, six mongrel dogs (12-16kg; 6-8mo old) were used. Twenty-four roots were replanted with either, (i) only periodontal defects (n=12; control group), or (ii) periodontal defects and PA-CM treatment (n=12; experimental group). In the experimental group, the PDL and cementum between notches was removed using a Gracey curette and soaked in 0.08mL water containing 80μg of a PA-CM for 2min. The dogs were killed at 4 and 8wk post-surgery. The invitro results showed that PA-CM stimulated the migration of PDL cells and promoted the differentiation and mineralization of PDL cells and cementoblasts. Real-time polymerase chain reaction analysis revealed stronger expression of Runx2, Osx, OC, Bsp and Cap mRNAs in the PA-CM-treated PDL cells and cementoblasts than those in the control cells. In vivo, newly formed PDL-like tissue and cementum-like tissue were observed partially between the root surfaces and newly formed bone in the experimental group. The regenerated PDL-like tissue in the experimental group was significantly higher than that in the control group at 8wk (p<0.05). The replacement resorption on the experimental group was significantly lower than that in the control group at 8wk (p<0.05). In addition, the amount of newly formed cementum-like tissue in the experimental group was significantly higher than that in the control group at 4 and 8wk (p<0.05). These results suggest that PA-CM has the potential to regenerate periodontal tissues in PDL and cementum defects.

Effects of the proteasome inhibitor, bortezomib, on cytodifferentiation and mineralization of periodontal ligament cells.

The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells. A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively. Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of β-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells. Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of β-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy.

Importance of CD44 in the proliferation and mineralization of periodontal ligament cells

CD44 is a transmembrane glycoprotein that exhibits various biological functions. Histological studies have shown that CD44 is expressed in periodontal ligament (PDL). We investigated the role of CD44 in the in vitro biological functions of PDL cells. Immunohistochemistry and immunocytochemistry were used to examine CD44 protein expression in mouse molars and human PDL cells, respectively. PDL cells isolated from human third molars were assayed for their proliferation and mineralization after stably silencing their expression of CD44 using the small hairpin RNA technique. An osteogenesis-associated quantitative polymerase chain reaction array was used to identify downstream molecules of CD44 signaling in osteogenic medium. The PDL cells, transduced with control small hairpin RNA, were used as controls in all assays. PDLs expressed CD44 in vitro and in vivo. When CD44 expression was stably suppressed in PDL cells, their proliferation and mineralization activities in both media were substantially decreased. The quantitative polymerase chain reaction array and Western blotting verified that bone morphogenetic protein-2 (BMP-2), fibroblast growth factor-1 (FGF-1) and intercellular adhesion molecule-1 (ICAM-1) were downregulated after CD44 was knocked down in PDL cells. Exogenous FGF-1 attenuated the proliferative suppression of CD44 knockdown cells. Exogenous BMP-2 rescued the suppressed mineralization of CD44 knockdown cells more than ICAM-1 did. The in vitro assays demonstrated that CD44 is crucial for the proliferation and mineralization of PDL cells and is possibly mediated by BMP-2, FGF-1 and ICAM-1. Further studies are required to verify the mediators and identify the physiological ligands of CD44 for possible application in periodontal regeneration.

Role of ferritin in the cytodifferentiation of periodontal ligament cells

This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

Mineralization effects of human platelet-rich plasma on human periodontium cells

To observe the effects of platelet-rich plasma (PRP) on inducing mineralization of human gingival fibroblasts (GF), periodontal ligament cells (PDLC) and alveolar bone osteoblasts (AOB). Human whole blood from healthy subjects was collected and PRP was obtained after twice centrifuged. GF, PDLC and AOB established from tissue explants were used at 4th passage in culture. All cells were seeded at density of 4 x 10(7)/L at 6 well tissue culture plate and cultured at standard condition for 96 hours, and then the different media was used. In the experimental group, the cells continued to be cultured in 50 ml/L PRP in DMEM media with mineral solution (10 mmol/L beta-glycerophosphate, 0.2 mmol/L freshly prepared ascorbic acid, 10 nmol/L dexamethasone), in the control group, the cells to be cultured in DMEM media with mineral solution, and in the blank group DMEM media was used. The media was changed every other day until day 30, the cells were dyed with von Kossa. Pictures were taken and analyzed by the image analysis software to semiquantitate the proportion of the positive area of von Kossa dye and attenuation aera of AOB. In PDLC and AOB, the quantity of mineralization nodule in the experimental group and the control group was more than that in the blank group (P < 0.05), and there were more mineralization nodules in the experimental group than in the control group (P < 0.05), while the amount of mineralization nodules in GF was low in the three groups (P > 0.05). Attenuation of AOB in the experimental group was less than the control group (P < 0.05). PRP can enhance the quantity of mineralization in PDLC and AOB in vitro and reduce attenuation in AOB, which suggests that PRP may be helpful to periodontal alveolar bone mineralization and regeneration.

Bone Sialoprotein Gene Transfer to Periodontal Ligament Cells May Not Be Sufficient to Promote Mineralization In Vitro or In Vivo

To improve regenerative therapies, it is important to understand the cells and factors modulating periodontal tissues. Our group has focused on bone sialoprotein (BSP), a mineralized tissue-selective protein considered to be involved in the initiation of cementogenesis and osteogenesis. In this study, we examined whether gene transfer of BSP into periodontal ligament (PDL) cells would result in an increased ability of PDL cells to promote mineralization in vitro and in vivo. PDL cells obtained from CD-1 mice were immortalized using simian virus (SV) 40 large T antigen (TAg) and designated SV-PDL cells. SV-PDL cells were infected in vitro with LacZ gene-expressing control adenovirus vector. A 1,000 plaque-forming unit (pfu) titer was selected (based on X-gal staining) and cells were infected with mouse BSP-expressing replication-deficient adenoviral vector to determine the mRNA expression and protein level of BSP. Total RNA was isolated from cells on days 2, 4, and 6. Media were obtained on days 3, 5, and 7 for protein determination. Northern blot analysis was performed for mRNA expression and Western blot analysis for protein expression. To test the effect of BSP gene transfer on the mineralization of PDL cells, in vitro (von Kossa) and in vivo (severe combined immunodeficiency [SCID] mice) experiments were performed. Under normal conditions, PDL cells do not express BSP transcripts and do not promote significant mineralization. SV-PDL cells infected with a BSP viral vector expressed and secreted substantial levels of BSP as confirmed by Northern and Western blot analysis. BSP mRNA and protein levels were strong on day 2 and still apparent on day 6, although not as great. However, no mineral nodule formation was noted either in vitro or in vivo. Although BSP is an important and necessary protein for mineralization, it may not be sufficient for promoting mineralization without the addition or removal of other factors. Further studies will help to clarify the specific factors required for promoting mineralization, a required step for designing predictable periodontal regenerative therapies.

Effect of Extract of Seeds of Carthamus tinctorius L. on Mineralization in Periodontal Ligament Cells and Osteoblastic Cells

Effect of Extract of Seeds of <i>Carthamus tinctorius L.</i> on Mineralization in Periodontal Ligament Cells and Osteoblastic Cells

Effects of the Isolated Extracts from Safflower Seeds on Mineralization of Periodontal Ligament Cells and Osteoblastic Cells

Effects of the Isolated Extracts from Safflower Seeds on Mineralization of Periodontal Ligament Cells and Osteoblastic Cells