HSPA1A is upregulated in periodontal ligament at early stage of tooth movement in rats

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

Differential expression of osteoblast and osteoclast chemmoatractants in compression and tension sides during orthodontic movement

Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading, and is supposed to be mediated by several host mediators, such as chemokines. In this study we investigated the pattern of mRNAs expression encoding for osteoblast and osteoclast related chemokines, and further correlated them with the profile of bone remodeling markers in palatal and buccal sides of tooth under orthodontic force, where tensile (T) and compressive (C) forces, respectively, predominate. Real-time PCR was performed with periodontal ligament mRNA from samples of T and C sides of human teeth submitted to rapid maxillary expansion, while periodontal ligament of normal teeth were used as controls. Results showed that both T and C sides exhibited significant higher expression of all targets when compared to controls. Comparing C and T sides, C side exhibited higher expression of MCP-1/CCL2, MIP-1α/CCL3 and RANKL, while T side presented higher expression of OCN. The expression of RANTES/CCL5 and SDF-1/CXCL12 was similar in C and T sides. Our data demonstrate a differential expression of chemokines in compressed and stretched PDL during orthodontic tooth movement, suggesting that chemokines pattern may contribute to the differential bone remodeling in response to orthodontic force through the establishment of distinct microenvironments in compression and tension sides.     

Strain-dependent up-regulation of ephrin-B2 protein in periodontal ligament fibroblasts contributes to osteogenesis during tooth movement

During orthodontic tooth movement, the application of adequate orthodontic forces allows teeth to be moved through the alveolar bone. These forces are transmitted through the periodontal ligaments (PDL) to the supporting alveolar bone and lead to deposition or resorption of bone, depending on whether the tissues are exposed to a tensile or compressive mechanical strain. Fibroblasts within the PDL (PDLF) are considered to be mechanoresponsive. The transduction mechanisms from mechanical loading of the PDLF to the initiation of bone remodeling are not clearly understood. Recently, members of the ephrin/Eph family have been shown to be involved in the regulation of bone homeostasis. For the first time, we demonstrate that PDLF exposed to tensile strain induce the expression of ephrin-B2 via a FAK-, Ras-, ERK1/2-, and SP1-dependent pathway. Osteoblasts of the alveolar bone stimulated with ephrin-B2 increased their osteoblastogenic gene expression and showed functional signs of osteoblastic differentiation. In a physiological setting, ephrin-B2-EphB4 signaling between PDLF and osteoblasts of the alveolar bone might contribute to osteogenesis at tension sites during orthodontic tooth movement.     

Histochemical examination of cathepsin K,MMP1 and MMP2 in compressed periodontal ligament during orthodontic tooth movement in periostin deficient mice

The purpose of this study was to investigate immunolocalization of collagenolytic enzymes including cathepsin K, matrix metalloproteinase (MMP) 1 and 2 in the compressed periodontal ligament (PDL) during orthodontic tooth movement using a periostin deficient (Pn-/-) mouse model. Twelve-week-old male mice homozygous for the disrupted periostin gene and their wild type (WT) littermates were used in these experiments. The tooth movement was performed according to Waldo’s method, in which elastic bands of 0.5 mm thickness were inserted between the first and second upper molars of mice under anesthesia. At 1 and 3 days after orthodontic force application, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the first molars and peripheral alveolar bones were extracted for histochemical analyses. Compared with WT mice, immunolocalization of cathepsin K, MMP1 and MMP2 was significantly decreased at 1 and 3 days after orthodontic tooth movement in the compressed PDL of Pn-/- mice, although MMP1-reactivity and MMP2-reactivity decreased at different amounts. Very little cathepsin K-immunoreactivity was observed in the assessed regions of Pn-/- mice, both before and after orthodontic force application. Furthermore, Pn-/- mice showed a much wider residual PDL than WT mice. Taken together, we concluded that periostin plays an essential role in the function of collagenolytic enzymes like cathepsin K, MMP1 and MMP2 in the compressed PDL after orthodontic force application.     

Periostin Associates with Notch1 Precursor to Maintain Notch1 Expression under a Stress Condition in Mouse Cells

Background

Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells.

Methods and Findings

In this study, however, we reported that periostin physically associates with the Notch1 precursor at its EGF repeats in the inside of cells. Moreover, by using the periodontal ligament of molar from periostin-deficient adult mice (Pn−/− molar PDL), which is a constitutively mechanically stressed tissue, we found that periostin maintained the site-1 cleaved 120-kDa transmembrane domain of Notch1 (N1™) level without regulating Notch1 mRNA expression. N1™ maintenance in vitro was also observed under such a stress condition as heat and H₂O₂ treatment in periostin overexpressed cells. Furthermore, we found that the expression of a downstream effector of Notch signaling, Bcl-xL was decreased in the Pn−/− molar PDL, and in the molar movement, cell death was enhanced in the pressure side of Pn−/− molar PDL.

Conclusion

These results suggest the possibility that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition, which is caused by its physical association with the Notch1 precursor.     

Immunohistochemical detection of matrix metalloproteinase-1 in the periodontal ligament of equine cheek teeth

The hypsodont equine cheek tooth erupts continuously throughout life. The collagen fibers of the periodontal ligament (PDL) have to remodel constantly to allow the tooth to move in an occlusal direction. Remodeling of the collagen fiber bundles needs to be well-coordinated in order to maintain functional tooth support. The aim of this study was to examine the role of matrix metalloproteinase-1 (MMP-1) in the collagen remodeling of the equine PDL under physiological conditions. Specimens containing the PDL interposed between the dental cementum and the alveolar bone were taken from nine Warmblood horses at three designated horizontal levels: subgingival, middle, and apical. The expression of MMP-1 was detected immunohistochemically. MMP-1 was found to be present in the specimens of all horses. Immunopositive fibroblasts/fibrocytes were accumulated within individual single collagen fascicles. Our results suggest that MMP-1 induced collagen degradation plays a central role in the physiological remodeling of the equine PDL. The distribution of MMP-1 positive fascicles indicates well-directed remodeling which occurs as an asynchronous process, so that only single collagen fascicles are remodeled at the same time. Due to this remodeling of one fascicle at a time, the overall anchorage of the tooth is preserved at all times.     

Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro

Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.This work was supported by a grant from the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).     

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway

BackgroundThe balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood.MethodsHere, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100 kpa static pressure for 1 h or 12 h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells.ResultsIn vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1 h relative to 12 h. In contrast, the expression ratio of RANKL/OPG was reduced at 1 h and significantly increased at 12 h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1 h.ConclusionsOur findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity.General significanceWe describe a novel potential mechanism related to tooth movement.     

The Biomechanical Function of Periodontal Ligament Fibres in Orthodontic Tooth Movement

Orthodontic tooth movement occurs as a result of resorption and formation of the alveolar bone due to an applied load, but the stimulus responsible for triggering orthodontic tooth movement remains the subject of debate. It has been suggested that the periodontal ligament (PDL) plays a key role. However, the mechanical function of the PDL in orthodontic tooth movement is not well understood as most mechanical models of the PDL to date have ignored the fibrous structure of the PDL. In this study we use finite element (FE) analysis to investigate the strains in the alveolar bone due to occlusal and orthodontic loads when PDL is modelled as a fibrous structure as compared to modelling PDL as a layer of solid material. The results show that the tension-only nature of the fibres essentially suspends the tooth in the tooth socket and their inclusion in FE models makes a significant difference to both the magnitude and distribution of strains produced in the surrounding bone. The results indicate that the PDL fibres have a very important role in load transfer between the teeth and alveolar bone and should be considered in FE studies investigating the biomechanics of orthodontic tooth movement.     

Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: Involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways

Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.     

Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration—culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.     

Finite element simulation of the behavior of the periodontal ligament: A validated nonlinear contact model

Due to its significance in tooth movement, the stress/deformation field of periodontium and the alveolar bone remodeling process, periodontal ligament (PDL) cannot be excluded from the studies investigating dental biomechanics regarding its excessive deformability. Therefore, many analytical and numerical researches are carried out to simulate its response and to create a constitutive model via experiments intending to discover the material properties of PDL. The aim of this study is to formulate a user specified contact model that can be used in conjunction with finite element (FE) software and reflects PDL’s influence on neighboring structures based on the currently available information, without requiring an actual volumetric finite element mesh of ligament. The results show good agreement with available experimental tooth mobility data. Smooth stress fields are obtained on the tooth root and alveolar bone, which is a significant aspect in bone-remodeling studies. The advantage of simulating PDL as a contact model at the interface of tooth root and the alveolar process instead of a solid-meshed FE model with poor geometric morphology and/or very dense mesh is expected to save pre/post-processing workforce, to increase the accuracy and to contribute to the smoothness of interface stress distributions.     

Development of a multipotent clonal human periodontal ligament cell line

Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage.     

Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar

One strategy evolved by teeth to avoid irreversible damage is to move and deform under the loads incurred during mastication. A key component in this regard is the periodontal ligament (PDL). The role of the bone underlying the PDL is less well defined. We study the interplay between the PDL and the underlying alveolar bone when loaded in the minipig. Using an Instron loading device we confirmed that the force-displacement curves of the molars and premolars of relatively fresh minipig intact mandibles are similar to those obtained for humans and other animals. We then used this information to obtain 3D images of the teeth before and after loading the tooth in a microCT such that the load applied is in the third linear part of the force displacement curve. We observed that at many locations there is a complimentary topography of the cementum and alveolar bone surface, strongly suggesting an active interplay between the tooth and the bone during mastication. We also observed that the loaded tooth does not come into direct contact with the underlying bone surface. A highly compressed layer of PDL is present between the tooth and the bone. The structure of the bone in the upper furcation region has a unique appearance with little obvious microstructure, abundant pores that have a large size range and at many locations the bone at the PDL interface has a needle-like shape. We conclude that there is a close interaction between the tooth, the PDL and the underlying alveolar bone during mastication. The highly compressed PDL layer that separates the tooth from the bone may fulfill a key shock absorbing function.     

Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study

Periodontal-ligament-derived cells (PDL cells) have stem-cell-like properties and, when implanted into periodontal defects in vivo, can induce periodontal regeneration including the formation of new bone, cementum, and periodontal ligament. We have previously demonstrated that PDL cell sheets, harvested from temperature-responsive cell culture dishes, have a great potential for periodontal regeneration. The purpose of this study has been to validate the safety and efficacy of human PDL (hPDL) cell sheets for use in clinical trials. hPDL tissues from three donors were enzymatically digested, and the obtained cells were cultured with media containing autologous serum in a cell-processing center (CPC). The safety and efficacy of hPDL cell sheets were evaluated both in vitro and in vivo. In vitro studies showed that the hPDL cell sheets had high alkaline phosphatase activity and periostin expression (known PDL markers) and no contamination with microorganisms. In vivo studies revealed that hPDL cell sheets, implanted with dentin blocks, induced the formation of cementum and PDL-like tissue in immunodeficient mice. The hPDL cells presented no evidence of malignant transformation. Thus, hPDL cell sheets created in CPCs are safe products and possess the potential to regenerate periodontal tissues.     

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Background

CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.

Results

In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.

Conclusion

These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.