PLAP-1/asporin inhibits activation of BMP receptor via its leucine-rich repeat motif

We previously identified the novel gene, periodontal ligament-associated protein-1 (PLAP-1)/asporin and reported that PLAP-1/asporin inhibited bone morphogenetic protein-2 (BMP-2)-induced cytodifferentiation of periodontal ligament (PDL) cells probably by direct interaction with BMP-2. Here, we elucidated the detailed regulatory mechanism of this protein on BMP-2-induced cytodifferentiation of PDL cells. Recombinant PLAP-1/asporin inhibited BMP-2-induced cytodifferentiation of PDL cells and competitively prevented BMP-2 from binding to the BMP receptor-IB (BMPR-IB), resulting in inhibition of BMP-dependent activation of Smad proteins. The induction of mutation to the leucine-rich repeat (LRR) motif, especially LRR5, within PLAP-1/asporin rescued the inhibitory effect of PLAP-1/asporin on BMP-2. By contrast, a 26-amino acid peptide in the PLAP-1/asporin LRR5 sequence inhibited BMP-2 activity. Our findings indicate that PLAP-1/asporin inhibits BMP-2-induced differentiation of PDL cells resulting from inactivation of the BMP-2 signaling pathway and that LRR, especially LRR5 of PLAP-1/asporin, plays an important role in the PLAP-1/asporin-BMP-2 interaction.     

Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

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.     

Extracellular role of S100A4 calcium-binding protein in the periodontal ligament

S100A4 is a member of the S100 calcium-binding protein family. S100A4 is expressed in several tissues; however, it is secreted by few cell types and its extracellular roles are unknown. In the present study we showed by in situ hybridization that periodontal ligament (PDL) cells express the S100A4 mRNA. Immunolocalization of the S100A4 protein in cryosections of PDL and analyses of PDL cell culture medium revealed that PDL cells secrete the S100A4 protein both in vivo and in vitro. Interestingly, addition of a recombinant mouse S100A4 protein to a bone marrow cell culture inhibited mineralized nodule formation in a concentration-dependent manner. This is the first report of an extracellular role for S100A4 as an inhibitor of mineralization. The PDL space is kept free of mineralization and S100A4 may be one of the factors responsible for such phenomenon.     

Future dentistry: cell therapy meets tooth and periodontal repair and regeneration

Cell-based tissue repair of the tooth and - tooth-supporting - periodontal ligament (PDL) is a new attractive approach that complements traditional restorative or surgical techniques for replacement of injured or pathologically damaged tissues. In such therapeutic approaches, stem cells and/or progenitor cells are manipulated in vitro and administered to patients as living and dynamic biological agents. In this review, we discuss the clonogenic potential of human dental and periodontal tissues such as the dental pulp and the PDL and their potential for tooth and periodontal repair and/or regeneration. We propose novel therapeutic approaches using stem cells or progenitor cells, which are targeted to regenerate the lost dental or periodontal tissue.     

The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement

A novel 90-kDa protein named periostin, which is preferentially expressed in the periosteum and the periodontal ligament (PDL), may play a role in bone metabolism and remodeling. However, the precise role of periostin in the PDL remains unclear. Therefore, we examined the expression of periostin mRNA during experimental tooth movement. Experimental tooth movement was achieved in 7-week-old male Sprague-Dawley rats. In control specimens without tooth movement, the expression of periostin mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The periostin mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The divergent expression of periostin mRNA in the PDL began to be observed at 3 h and continued up to 96 h after tooth movement. The maximum changes, which showed stronger staining in the pressure sites than in the tension sites, were observed at 24 h. The expression of periostin mRNA in the PDL 168 h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggest that periostin is one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.     

Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo

The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering-based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T-antigen and hTERT (Cell Tissue Res 2006; 324: 117-125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1-4, 1-11, and 1-24) that express RUNX-2, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and alpha-SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1-4 and 1-11 and that STRO-1 was expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 differentiated into osteoblastic cells and adipocytes when cultured in lineage-specific differentiation media. Four weeks after transplanting cell line 1-11 into immunodeficient mice with beta-tricalcium phosphate (beta-TCP), the transplant produced cementum/bone-like tissues around the beta-TCP. Eight weeks after transplantation, the 1-11 cell transplant formed PDL-like structures on the surface of the beta-TCP. These data suggest that cell line 1-11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium.     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Identification of genes preferentially expressed in periodontal ligament: specific expression of a novel secreted protein, FDC-SP

Gene expression in human periodontal ligament (PDL) was examined by suppression subtractive hybridization to identify genes that are preferentially expressed in tissue compared to cultured PDL fibroblasts. The most enriched genes in a subtracted cDNA library are primarily genes for extracellular matrix components, types I and III collagen, lumican, periostin, and asporin, among others, whose expression conveys unique mechanical properties to the PDL. Also within this group is the gene for follicular dendritic cell secreted protein (FDC-SP), a small protein like statherin in saliva, not previously found in PDL. FDC-SP's presence in PDL was confirmed by in situ hybridization in mouse which also showed that it was definitely present in the parotid gland, but, surprisingly, not in the other salivary glands: submandibular and sublingual. Since only normal tissue was examined, these findings suggest that FDC-SP plays an important but previously unsuspected role within oral connective tissue.     

Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression

Background  

Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.     

Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig's epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways.     

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.