Periodontal Ligament-associated Protein-1 Research Articles

Mice Lacking PLAP-1/Asporin Show Alteration of Periodontal Ligament Structures and Acceleration of Bone Loss in Periodontitis.

Periodontal ligament-associated protein 1 (PLAP-1), also known as Asporin, is an extracellular matrix protein expressed in the periodontal ligament and plays a crucial role in periodontal tissue homeostasis. Our previous research demonstrated that PLAP-1 may inhibit TLR2/4-mediated inflammatory responses, thereby exerting a protective function against periodontitis. However, the precise roles of PLAP-1 in the periodontal ligament (PDL) and its relationship to periodontitis have not been fully explored. In this study, we employed PLAP-1 knockout mice to investigate its roles and contributions to PDL tissue and function in a ligature-induced periodontitis model. Mandibular bone samples were collected from 10-week-old male C57BL/6 (WT) and PLAP-1 knockout (KO) mice. These samples were analyzed through micro-computed tomography (μCT) scanning, hematoxylin and eosin (HE) staining, picrosirius red staining, and fluorescence immunostaining using antibodies targeting extracellular matrix proteins. Additionally, the structure of the PDL collagen fibrils was examined using transmission electron microscopy (TEM). We also conducted tooth extraction and ligature-induced periodontitis models using both wild-type and PLAP-1 KO mice. PLAP-1 KO mice did not exhibit any changes in alveolar bone resorption up to the age of 10 weeks, but they did display an enlarged PDL space, as confirmed by μCT and histological analyses. Fluorescence immunostaining revealed increased expression of extracellular matrix proteins, including Col3, BGN, and DCN, in the PDL tissues of PLAP-1 KO mice. TEM analysis demonstrated an increase in collagen diameter within the PDL of PLAP-1 KO mice. In line with these findings, the maximum stress required for tooth extraction was significantly lower in PLAP-1 KO mice in the tooth extraction model compared to WT mice (13.89 N ± 1.34 and 16.51 N ± 1.31, respectively). In the ligature-induced periodontitis model, PLAP-1 knockout resulted in highly severe alveolar bone resorption, with a higher number of collagen fiber bundle tears and significantly more osteoclasts in the periodontium. Our results demonstrate that mice lacking PLAP-1/Asporin show alteration of periodontal ligament structures and acceleration of bone loss in periodontitis. This underscores the significant role of PLAP-1 in maintaining collagen fibrils in the PDL and suggests the potential of PLAP-1 as a therapeutic target for periodontal diseases.

Periodontal ligament-associated protein-1 promotes osteoclastogenesis in mice by modulating TGF-β1/Smad1 pathway.

Periodontal ligament-associated protein-1 (PLAP-1), an important target molecule of osteoarthritis research, may affect alveolar bone resorption. The aim of our study was to comprehensively and systematically detect the effect of PLAP-1 on alveolar bone resorption and the underlying mechanism in PLAP-1 knockout mouse models. We used a PLAP-1 knockout (C57BL/6N-Plap-1-/- ) mouse model to investigate the effect of PLAP-1 on osteoclast differentiation and the underlying mechanism by adding Porphyromonas gingivalis lipopolysaccharide to stimulate bone marrow-derived macrophages. The effect of PLAP-1 on alveolar bone resorption and the underlying mechanism were studied using a ligature periodontitis model, with microcomputed tomography imaging, immunochemistry, and immunofluorescence. The in vitro analysis results demonstrated that PLAP-1 knockout significantly inhibited osteoclast differentiation under both normal and inflammatory conditions. Bioinformatic analysis, immunofluorescence, and co-immunoprecipitation showed colocalization and interaction between PLAP-1 and transforming growth factor beta 1 (TGF-β1). The phosphorylation of Smad1 was reduced in the PLAP-1 knockout cells compared with that in the cells from wild-type mice. The in vivo analysis results demonstrated that PLAP-1 knockout decreased bone resorption and the levels of osteoclast differentiation markers in experimental periodontitis compared with those in wild-type mice. Immunofluorescence staining confirmed colocalization of PLAP-1 and TGF-β1 in the experimental periodontitis model. The phosphorylation level of Smad1 was significantly reduced in PLAP-1 knockout mice compared with that in wild-type mice. This study revealed that the knockout of PLAP-1 inhibits osteoclast differentiation and decreases alveolar bone resorption through the TGF-β1/Smad1 signaling pathway, which could serve as an innovative target for the prevention and treatment of periodontitis.

Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway.

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-β1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.

Periodontal ligament-associated protein-1 engages in teeth overeruption and periodontal fiber disorder following occlusal hypofunction.

Teeth overeruption is a problem of clinical significance, but the underlying mechanism how changes in external occlusal force convert to the periodontium remodeling signals has been a largely under explored domain. And recently, periodontal ligament-associated protein-1 (PLAP-1)/asporin was found to play a pivotal role in maintaining periodontal homeostasis. The aim of this study was to explore the function of PLAP-1 in the periodontally hypofunctional tissue turnover. After extracting left maxillary molars in mice, the left and right mandibular molars were distributed into hypofunction group (HG) and control group (CG), respectively. Mice were sacrificed for radiographic, histological, and molecular biological analyses after 1, 4 and 12 weeks. In vitro, dynamic compression was applied using Flexcell FX-5000 Compression System to simulate intermittent occlusal force. The expression of PLAP1 in loaded and unloaded human periodontal ligament cells (hPDLCs) was compared, and its molecular biological effects were further explored by small interfering RNA (siRNA) targeting PLAP1. In vivo, fiber disorder in periodontal ligament (PDL), bone apposition at furcation regions, and bone resorption in alveolar bone were illustrated in the HG compared with the CG. In addition, PLAP-1 positive area decreased significantly in PDL following occlusal unloading. In vitro, the loss of compressive loading relatively downregulated PLAP1 expression, which was essential to promote collagen I but inhibit osterix and osteocalcin expression in hPDLCs. PLAP-1 presumably plays a pivotal role in occlusal force-regulated periodontal homeostasis by facilitating collagen fiber synthesis in hPDLCs and suppressing excessive osteoblast differentiation, further preventing teeth from overeruption. Further evidence in PLAP-1 conditional knockout mice is needed.

Reciprocal role of PLAP-1 in HIF-1α-mediated responses to hypoxia.

To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1α activity and periodontal ligament-associated protein-1 (PLAP-1) expression in human periodontal ligament cells (HPDLs). Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF-1α. PLAP-1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP-1 expression has been demonstrated in hypoxic regions, the involvement of PLAP-1 in responses to hypoxia has not been revealed. HPDLs were cultured under normoxic (20% O2 ) or hypoxic (1% O2 ) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1α were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)-containing luciferase vector in the presence or absence of recombinant PLAP-1 or PLAP-1gene transfection. Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP-1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP-1 expression in HPDLs. Hypoxia-induced PLAP-1 expression was significantly suppressed in the presence of chetomin. PLAP-1-suppressed HPDLs showed increased HIF-1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP-1. In the presence of recombinant PLAP-1, hypoxia-induced HRE activity of 293T cells was significantly suppressed in a dose-dependent manner. Transfection of the PLAP-1gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions. PLAP-1 expression is upregulated under hypoxic conditions through HIF-1α activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1α signaling.

Mice lacking PLAP-1/asporin counteracts high fat diet-induced metabolic disorder and alveolar bone loss by controlling adipose tissue expansion

Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.

LncRNA DCST1-AS1 inhibits PDLCs' proliferation in periodontitis and may bind with miR-21 precursor to upregulate PLAP-1.

This study aimed to investigate the potential interactions among long noncoding RNA domain containing 1-antisense (lncRNA DCST1-AS1), miR-21, and periodontal ligament-associated protein-1 (PLAP-1) in periodontitis. It has been verified that miR-21 can target PLAP-1 to regulate the osteogenic differentiation of periodontal ligament cells (PDLCs). Differential expression of DCST1-AS1 and miR-21 in PDLCs derived from periodontitis patients and healthy controls was determined by qPCR and unpaired t test. QPCR and Western blots were conducted to evaluate the effects of overexpression of DCST1-AS1 and miR-21 on the expression of PLAP-1. CCK-8 assay was applied to evaluate the effect of DCST1-ASI, miR-21, or PLAP-1 on PDLCs' proliferation. Western blotting was conducted to detect the expression levels of CKD family (CDK4, CDK6, and CCND1). DCST1-AS1 was downregulated in PDLCs derived from periodontitis patients, and its expression was inversely correlated with the expression of miR-2 but positively correlated with PLAP-1. Bioinformatics analysis showed that DCST1-AS1 might bind with miR-21 precursor but not mature miR-21. Transfection experiments showed that overexpression of DCST1-AS1 led to decreased expression levels of miR-21 and significantly increased the expression levels of PLAP-1 at both mRNA and protein levels, while overexpression of miR-21 resulted in a dramatic lower level of PLAP-1. CCK-8 assay indicated that overexpression of DCST1-AS1 or PLAP-1 prohibited PDLCs' proliferation. However, elevation of miR-21 had a contrary effect on the proliferation of PDLCs. And increased expression levels of DCST1-AS1 could significantly inhibit the expression of CDK4, CDK6, and CCND-1, while overexpression of miR-21 inversed the effects of DCST1-AS1. Therefore, the expression levels of DCST1-AS1 are much lower in periodontitis patients compared to that in healthy controls, and overexpression of DCST1-AS1 can significantly elevate the expression of PLAP-1 by inhibiting miR-21 in PDLCs.

Identification of a New Theca/Interstitial Cell-Specific Gene and Its Biological Role in Growth of Mouse Ovarian Follicles at the Gonadotropin-Independent Stage.

Theca/interstitial cells are responsible for the growth and maturation of ovarian follicles. However, little is known about the theca/interstitial cell-specific genes and their functions. In this study, we explored transcriptomes of theca/interstitial cells by RNA-seq, and the novel biological roles of a theca cell marker, asporin (Aspn)/periodontal ligament-associated protein 1 (PLAP-1). RNA-seq detected 432 and 62 genes expressed specifically in theca/interstitial cells and granulosa cells isolated from 3-weeks old mouse ovaries. Gene ontology analysis demonstrated that these genes were largely categorized into four major groups: extracellular matrix organization-related terms, chemotaxis-related terms, the angiogenesis-related terms, and morphogenesis-related terms. In situ hybridization demonstrated that the newly detected representative gene, Aspn/PLAP-1, was detected specifically in the outer layer of theca cells in contrast with the expression of the basal lamina-specific gene, Nidgen-1. Intriguingly, an Aspn/PLAP-1 antibody completely arrested the growth of secondary follicles that is the gonadotropin-independent follicle developmental stage. Furthermore, transforming growth factor-β (TGF-β)-triggered signaling was induced by the Aspn/PLAP-1 antibody treatment, which is consistent with the inhibitory effect of Aspn/PLAP-1 on TGF-β. Altogether, these results suggest that theca cells are classified into subpopulations on the basis of new marker genes and their biological functions, and provide evidence that Aspn/PLAP-1 is expressed exclusively in the outer layer of theca cells and plays a pivotal role in the growth of secondary follicles via downregulation of the canonical TGF-β signaling cascade.

Self-Assembly of an Organized Cementum-Periodontal Ligament-Like Complex Using Scaffold-Free Tissue Engineering.

A major challenge in regenerating periodontal tissues is emulating its complex structure containing both mineralized and soft tissues. In this study, scaffold-free tissue constructs engineered using periodontal ligament cells (PDLCs), which contain a population of adult stem/progenitor cells, self-assembled into an organized multi-tissue structure comprising a mineralized cementum-like core enclosed within a periodontal ligament (PDL)-like tissue. Scaffold-free engineered constructs were formed by culturing human PDLCs to form a cell sheet on six-well dishes containing two minutien pins placed 7 mm apart. The cell sheet was contracted by the cells to roll into the pins forming a cylindrical construct anchored on either end by the pins. These tissues were approximately 1 mm in diameter and 7 mm long and contained only the cells and their endogenous matrix. These scaffold-free engineered constructs exhibited two structurally distinct tissues, one in the center of the construct and another on the periphery. The center tissue was mineralized and expressed alkaline phosphatase and bone sialoprotein, similar to cementum. The peripheral tissue was not calcified and expressed periodontal ligament-associated protein-1 and periostin, which is characteristic of the periodontal ligament. This tissue organization was seen after in vitro culture and maintained in vivo following subcutaneous implantation in immunocompromised mice. These data demonstrate that scaffold-free tissue engineering facilitates PDLCs to self-assemble into an organized cementum-PDL-like complex. These engineered tissues could be used as implantable grafts to regenerate damaged periodontal tissues or as model systems to study PDLC biology and mechanisms driving organized tissue assembly within the periodontium.

Periodontal ligament-associated protein-1 gets involved in the development of osseous eruption canal.

Osseous eruption is an important stage of tooth eruption process. The role of periodontal ligament-associated protein-1 (PLAP-1/asporin) in the development of osseous eruption canal remain undefined and were the focus of this study. C57BL/6 mice at postnatal days P11-13 and P 15-16 were chosen. The development of osseous eruption canal of lower first molar was observed and osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP). PLAP-1 expression in the process of osseous eruption (OE, P11-13) and post- osseous eruption (P-OE, P15-16) was assessed by immunohistochemistry, immunofluorescence and western blotting. Receptor activator of NF-κB ligand (RANKL) distribution in the process was also assessed by immunohistochemistry. A double immunofluorescence stain was used to reveal PLAP-1 in association with CD68 (osteoclast maker). Fresh occlusal tissues of erupting lower first molars at OE and P-OE were separated to detected RANKL/OPG ratio by western blotting to elucidate related mechanisms. At osseous eruption (OE), osseous and mucosal tissues could be observed on the occlusal side of lower first molar. Osseous eruption canal was developing. Many osteoclasts were found around occlusal alveolar bone in the development of osseous eruption canal. At post- osseous eruption (P-OE), osseous eruption canal had been built, only mucosal tissues were observed, and few osteoclasts were detected. More PLAP-1 expression was detected at OE, compared with that at P-OE. Similar distributions of PLAP-1 and RANKL in occlusal bone tissues of erupting lower first molars were detected at OE. Colocalization of PLAP-1 and CD68 revealed the positive relationship between PLAP-1 and osteoclasts in the development of osseous eruption canal. PLAP-1 positively correlated with RANKL and CD68+ osteoclasts, and areas of bone resorption. Higher RANKL/OPG ratio was detected at OE, compared with that at P-OE. PLAP-1 gets involved in the development of osseous eruption canal.

Periodontal ligament-associated protein-1 gets involved in experimental periodontitis.

Periodontalligament-associated protein-1 (PLAP-1) is an important regulator of osteogenic differentiation ofperiodontalligament cells and plays important role in the homeostasis of periodontal tissues. But the role of PLAP-1 in periodontitis is poorly understood. Expressions of PLAP-1 in experimental periodontitis are observed to elucidate whether PLAP-1 gets involved in the pathogenesis of periodontitis. Wistar rats were randomly allocated to two groups (n=6/group): Ligation group and Control group. PLAP-1 expression in experimental periodontitis was assessed by immunohistochemistry and collagen fibers in periodontal ligament were observed using picrosirius red staining. Expressions of PLAP-1 and CD68 in periodontitis were colocalized by double-labelled immunofluorescence. To further examine the relationship between PLAP-1 and osteoclastogenesis in experimental periodontitis, acute periodontal inflammatory infiltration and alveolar bone destruction were induced by administering ligated rats with 10ng/mL tumor necrosis factor alpha (TNF-α; ligation+TNF-α group, n=6). Alveolar bone loss was observed by micro-computed tomography (Micro-CT), and osteoclasts were identified by tartrate-resistant acid phosphatase staining (TRAP). Expressions of PLAP-1 in TNF-α stimulated human periodontal ligament cells were also detected at 24 and 48hours by western blotting. PLAP-1 expression levels in periodontal ligament cells and collagen fibers were lower in the ligation group,compared with the control group. Similarly, TNF-α decreased PLAP-1 expression in human periodontal ligament cells in vitro. Degradation or destruction of collagen fibers accompanied the reduced PLAP-1 expression in the periodontal ligament in the ligation group. Colocalization of PLAP-1 and CD68 revealedthe positive relationship between PLAP-1 and CD68+ infiltrating cells in periodontitis. More PLAP-1-positive inflammatory cells were found in the ligation+TNF-α group, compared with the ligation+saline group. PLAP-1-positive inflammatory cells are involved in the pathogenesis of periodontitis. An increase in PLAP-1-positive inflammatory cell number contributes periodontal inflammation and alveolar bone loss.

Periodontal ligament‑associated protein‑1 delays rat periodontal bone defect repair by regulating osteogenic differentiation of bone marrow stromal cells and osteoclast activation.

The aim of the present study was to assess the roles of periodontal ligament‑associated protein‑1(PLAP‑1) in the osteogenic differentiation of rat bone marrow stromal cells(rBMSCs) and in osteoclast activation during the repair of rat periodontal bone defects. Male, 6‑week‑old, Wistar rats treated with periodontal bone defects were randomly assigned to 3groups: The PLAP‑1‑transfected rBMSC group(PLAP‑1group), the empty vector‑transfected rBMSC group(vectorgroup) and the normal rBMSCgroup (control group). Specimens were obtained at 2, 4 and6weeks post‑surgery. Histological observation and micro‑computed tomography were applied to evaluate the repair effect. The bone defect areas of the mandible were dissected for western blotting and reverse transcription-quantitative polymerase chain reaction(RT‑qPCR). Osteogenesis‑associated proteins, including alkaline phosphatase(ALP), bone sialoprotein(BSP), runt-related transcription factor 2 (Runx2), Osterix(Osx) and osteocalcin(OC), as indicators of rBMSC‑induced osteogenesis, were examined by RT-qPCR and western blotting. Osteoclasts were identified and quantified using tartrate‑resistant acid phosphatase staining. Meanwhile, the receptor activator of nuclear factor κΒ ligand (RANKL)/οsteoprotegerin (OPG) ratio was quantified to assess osteoclast activation by western blotting. Τhe repair effect of the PLAP‑1group was significantly worse than that of the vector and control groups. In the PLAP‑1group, newly formed and mineralized bones were significantly less in quantity than that in the other two groups(P<0.05), and the expression of osteogenic proteins(ALP, BSP, Runx2, Osx and OC) was also reduced(P<0.01). However, there was no significant difference between the vector and control groups. The RANKL/OPGratio was upregulated in the PLAP‑1group due to decreased OPG protein expression and a simultaneous increase in RANKL protein expression(P<0.01), and more osteoclasts were activated in the PLAP‑1group(P<0.01). In conclusion, the present study found that PLAP‑1 delays rat periodontal bone defect repair by inhibiting osteogenic differentiation and promoting osteoclast activation, mainly dependent on the upregulation of the RANKL/OPG ratio.

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 Specific Differentiation of Dental Follicle Stem Cells into Osteoblast, Fibroblast, and Cementoblast.

The dental follicle is a source of dental follicle stem cells (DFCs), which have the potential to differentiate into the periodontal lineage. DFCs therefore are of value in dental tissue engineering. The purpose of this study was to evaluate the effect of growth factor type and concentration on DFC differentiation into periodontal specific lineages. DFCs were isolated from the human dental follicle and characterized for the expression of mesenchymal markers. The cells were positive for CD-73, CD-44, and CD-90; and negative for CD-33, CD-34, and CD-45. The expression of CD-29 and CD-31 was almost negligible. The cells also expressed periodontal ligament and cementum markers such as periodontal ligament-associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and cementum protein-1 (CEMP-1), however, the expression of osteoblast markers was absent. Further, the DFCs were cultured in three different induction medium to analyze the osteoblastic, fibroblastic, and cementoblastic differentiation. Runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) activity, alizarin staining, calcium quantification, collagen type-1 (Col-1), and osteopontin (OPN) expression confirmed the osteoblastic differentiation of DFCs. DFCs cultured in recombinant human FGF-2 (rhFGF-2) containing medium showed enhanced PLAP-1, FGF-2, and COL-1 expression with increasing concentration of rhFGF-2 which thereby confirmed periodontal ligament fibroblastic differentiation. Similarly, DFCs cultured in recombinant human cementum protein-1 (rhCEMP-1) containing medium showed enhanced bone sialoprotein-2 (BSP-2), CEMP-1, and COL-1 expression with respect to rhCEMP-1 which confirmed cementoblastic differentiation. The expression of osteoblast, fibroblast, and cementoblast-related genes of DFCs cultured in induction medium was enhanced in comparison to DFCs cultured in noninduction medium. Thus, growth factor-dependent differentiation of DFCs into periodontal specific lineages was proved by quantitative analysis.

Overexpression of PLAP-1 in bone marrow stromal cells inhibits the rat critical-size skull defect repair.

Periodontal ligament-associated protein-1 (PLAP-1) is an osteogenisis negative regulatory gene that inhibits the differentiation of rat bone marrow stromal cells (rBMSCs) into osteoblasts in vitro. The aim of this study was to verify whether PLAP-1 acted as an inhibitory factor of rBMSCs in rat critical-size skull defect repair in vivo. Six-week old male Wistar rats treated with a drill-hole injury were randomly assigned into five groups PLAP-1-transfected rBMSC group, empty vector-transfected rBMSC group, normal rBMSC group, collagen group and blank control group according to the treatment factors. Skull specimens were obtained at 8 weeks after surgery. X-ray examination, histological observation of hard tissue slices (HE, Masson staining and von Kossa staining), immunohistochemical staining were applied to evaluate the repair effects. X-ray examination showed that repair effect of the PLAP-1-transfected rBMSC group was significantly worse than that of the empty vector-transfected rBMSC group and normal rBMSC group. In HE staining, regenerated bone could only be observed in the partial defect area of the PLAP-1-transfected rBMSC group. However, new bone filled nearly the entire defects in the empty vector-transfected rBMSC group and normal rBMSC group. Mineralization of new bone in the two groups was significantly higher than that of the PLAP-1-transfected rBMSC group, according to Masson and von Kossa staining. Meanwhile, the transfected PLAP-1 gene worked well in vivo. Positive expression of PLAP-1 protein was only distributed in the newly formed bone of the PLAP-1-transfected rBMSC group, no positive staining was observed in the other four groups. Overexpression of PLAP-1 in rBMSCs inhibits new bone formation and mineralization in rat critical-size skull defects in vivo.

Overexpression of the PLAP-1 gene inhibits the differentiation of BMSCs into osteoblast-like cells.

Periodontal ligament-associated protein-1 (PLAP-1) is a newly discovered member of the extracellular matrix family of proteins known as proteoglycans and is a negative regulator that plays a crucial role in the homeostasis of periodontal tissues. It can protect the periodontal ligament from excessive osteogenesis. However, the molecular mechanisms of PLAP-1 during osteogenic differentiation and osteogenesis remain unclear. In this study, we constructed a PLAP-1 recombinant retroviral plasmid vector named pBABE-hygro-PLAP-1. We transfected this plasmid into rat bone marrow stromal cells (rBMSCs) to obtain a stable cell line with overexpression of PLAP-1 to verify whether PLAP-1 also acts as an inhibitory factor in rBMSCs during bone mineralization. A rBMSC line stably overexpressing PLAP-1 was established successfully as determined by the mRNA levels of PLAP-1, which were measured by real time-qPCR (RT-qPCR), and protein expression, which was measured by immunocytochemistry and western blot analysis. At the same time, a Cell Counting Kit-8 assay did not reveal any statistically significant changes in the transfected cells (P > 0.05). Then, mineral-inducing cultures were performed, and mineralized nodules were observed at weeks 2, 3 and 4 under a microscope. Alizarin Red (Sigma) staining was performed at 4 week to illustrate calcium accumulation. The mineralized nodules in the PLAP-1-transfected rBMSC group were fewer than those in the control groups. The time span of the formation of the mineralized nodules was prolonged. Meanwhile, osteogenic genes were also detected in the mineral-inducing cells by RT-qPCR. An RT-qPCR analysis demonstrated that the levels of the osteoblast markers of rBMSCs that were transfected with pBABE-hygro-PLAP-1, including Runx2, Osterix, alkaline phosphatase, bone sialoprotein and osteocalcin, were lower than those in the non-transfected rBMSCs and rBMSCs that were transfected with empty vector (P < 0.01). These results suggest that PLAP-1 has an inhibitory function in rBMSCs when they differentiate into osteoblast-like cells.

Effect of orthodontic force on expression of Asporin after tooth auto-transplantation: an experimental study in Beagle dogs

To evaluate the effect of orthodontic light force on the expression of Asporin, bone morphogenetic proteins-2 (BMP-2) and alkaline phosphatase (ALP) after auto-transplantation. Thirty-two maxillary and mandibular incisors in four 13 month-old male Beagle dogs were auto-transplanted to the other side of the same jaw. The teeth were all endodontically treated and divided into four groups, control (group 1) and three experimental groups (groups 2, 3 and 4).In control group, the teeth were unloaded. In the other three experimental groups, continuous force was applied in the 1st week (group 2), 2nd week (group 3) and 4th week (group 4) after auto-transplantation, respectively. The dogs were sacrificed in the 8th week. The mRNA expressions of Asporin,BMP-2 and ALP were examined by real time PCR. The expression of periodontal ligament associated protein-1 (PLAP-1) was examined by Western blotting. The data were statistically analyzed using SPSS 17.0 and one-way analysis of variance (ANOVA). In group 3, the expression of Asporin mRNA (2.047 ± 0.281) was higher than that in the other three groups, while the expression of BMP-2 (1.358 ± 0.095) was lower than that in group 2 and control group (P < 0.05). The expression of PLAP-1 (1.054 ± 0.113) in group 3 was higher than other groups, while significant difference was found between any two groups. Orthodontic force could stimulate the expression of Asporin and PLAP-1. The orthodontic force applied in the 2nd week after the tooth auto-transplantation, the expression of Asporin and PLAP-1 reached the highest level.

In vitro human periodontal ligament-like tissue formation with porous poly-l-lactide matrix

This study aimed to establish an in vitro human periodontal ligament-like tissue (HPdLLT) by three-dimensional culturing of human periodontal ligament fibroblasts (HPdLFs) in a porous poly-l-lactide (PLLA) matrix modified hydrophilically with ammonia solution. After ammonia modification, the surface roughness and culture-medium-soaking-up ability of the PLLA matrix increased, whereas the contact angle of water drops decreased. The thickness, porosity, and pore size of the PLLA matrix were 400±50μm, 83.3%, and 75–150μm, respectively. HPdLFs (1×105 cells) were seeded on the modified PLLA matrix and centrifuged to facilitate seeding into its interior and cultured for 14days. Scanning electron microscope (SEM) observation, proliferation assay, picrosirius-red staining, and real-time polymerase chain reaction (RT-PCR) for type-1 collagen (COL1), periodontal ligament associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and alkaline phosphatase (ALP) mRNA were conducted on days 1, 3, 7, and 14. HPdLFs were observed entirely from the surface to the rear side of the matrix. Cell proliferation analysis, SEM observation, and picrosirius-red staining showed both progressive growth of 3D-cultured HPdLFs and extracellular matrix maturation by the secretion of COL1 and type 3 collagen (COL3) from days 1 to 14. Expressions of COL1, PLAP-1, and FGF-2 mRNA suggested the formation of cellular components and supplementation of extracellular components. Expressions of ALP, COL1, and PLAP-1 mRNA suggested the osteogenic potential of the HPdLLT. The results indicated in vitro HPdLLT formation, and it could be used in future periodontal ligament tissue engineering to achieve optimal periodontal regeneration.

MiR-21 and miR-101 regulate PLAP-1 expression in periodontal ligament cells

Periodontal ligament-associated protein-1 (PLAP-1/asporin) is a special marker in periodontal ligament tissue. It is an important regulator of osteogenic differentiation of periodontal ligament cells (PDLCs). This marker is also a prerequisite for periodontal ligament development and mineralization in maintaining homeostasis of the periodontium. However, the molecular mechanisms of the regulation of PLAP-1 expression at the post-transcriptional level remain unknown. By contrast, microRNAs (miRNAs) provide an additional level of regulation beyond that of transcription factors via regulation of the post-transcriptional control of gene expression. This study was designed to analyze miRNA differential expression patterns of PDLCs at various osteoblastic differentiation stages and to determine the contribution of miRNAs in the regulation of PLAP-1 expression during osteoblast differentiation. Bioinformatic analysis was performed to predict miRNAs that potentially regulate the gene expression of PLAP-1. Dual luciferase reporter assay and qRT-PCR were performed to confirm the effects of these miRNAs on PLAP-1 gene expression. Our results indicated that mir-101 and mir-21 target PLAP-1 to regulate its expression during osteogenic differentiation of PDLCs.

Expression of periodontal ligament-associated protein-1 in normal periodontal tissues and cells in rat

To examine the expression of periodontal ligament-associated protein-1 (PLAP-1) in the periodontal tissues and periodontal ligament cells (PDLC). The PLAP-1 expression in normal periodontal tissue was examined by immunohistochemistry. The protein expression and mRNA transcription of PLAP-1 in PDLC were investigated by immunocytochemistry and reverse transcription-polymerase chain reaction. PLAP-1 was expressed in periodontium but not in cementum, alveolar bone and gingival tissues. PLAP-1 expression was observed in cell plasma, but not in nuclei. There was a 350 bp electrophoresis band representing PLAP-1 mRNA. PLAP-1 may play a role in physiology of periodontal tissues and cells in normal adult rats.