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??Sclerostin/SOST??mainly expressed in osteocytes??is a negative regulator of bone formation and is regulated by mechanical stimulus??which plays an important role in orthodontic tooth movement. Wnt and BMP are two important signaling pathways in bone metabolic regulation. SOST can regulate osteoblastic differentiation and bone formation by binding type ??or??receptors and co-receptor LRP5/6 to inhibit BMP and Wnt signaling pathways. This review covers the effect of Sclerostin/SOST on orthodontic tooth movement after mechanical loading and its mechanism??and the clinical significance as well as prospect of Sclerostin/SOST in clinical application.     

Calcitonin induces collagen synthesis and osteoblastic differentiation in human periodontal ligament fibroblasts

BackgroundExtracellular matrix (ECM) secretion and osteogenic differentiation in periodontal ligament fibroblasts (PDLF) facilitate the neogenesis of alveolar bone, which is the cellular basis for alveolar bone repair. Calcitonin (CT) has been reported to play an important role in promoting ECM expression and inducing osteogenic differentiation in osteoblast, but its effects on PDLFs remain obscure.MethodsThe expression of CT, transforming growth factor-beta 1(TGF-β1) and bone morphogenetic protein (BMP) in gingival crevicular fluid (GCF) was measured by ELISA. The effects of CT on collagen synthesis and osteogenic differentiation in hPDLFs were investigated by using the primarily cultured hPDLFs infected with adenovirus carrying the CT gene. Gene expression was measured by quantitative PCR and western blot.ResultsThe expression of CT in gingival crevicular fluid (GCF) of patients with periodontitis was significantly higher than that of healthy subjects. In addition, CT expression correlated with the clinical indexes including probing pocket depth (PPD), clinical attachment level (CAL), and gingival index (GI). The in vitro study demonstrated that overexpression of CT by adenovirus infection increased the expression of TGF-β1, collagen type I and III, and osteoblastic markers including BMP-2/-4, alkaline phosphatase and osteocalcin in human PDLFs. Moreover, CT-enhanced collagen synthesis was abrogated in hPDLFs transfected with TGF-β1 siRNA, and CT-induced osteoblastic differentiation was blocked in hPDLFs by BMPs inhibitor noggin.ConclusionsThese results suggest that CT promotes collagen synthesis and osteogenic differentiation in hPDLFs via the TGF-β1 and BMPs signaling pathways, respectively.     

Stem Cell Regulatory Gene Expression in Human Adult Dental Pulp and Periodontal Ligament Cells Undergoing Odontogenic/Osteogenic Differentiation

IntroductionDuring development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes.MethodsIn this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation.ResultsOur results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration.ConclusionsThis study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.     

Effects of two vitamins,two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1 cells

The purpose of this investigation was to examine the effects of the addition of five cytokines such as vitamin C, vitamin D, bone morphogenetic protein (BMP), transforming growth factor-beta (TGF-beta) and dexamethasone (Dex) to Dulbecco's modified Eagle (DME) medium on the proliferation of Sprague-Dawley (SD) rats' bone marrow stromal cells and osteoblastic MC3T3-E1 cells by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. For bone marrow stromal cells, vitamin C was needed for doubling the cell viability. While BMP, TGF-beta and vitamin D maintained the growth rate given by vitamin C, Dex with beta-glycerophosphate (beta-GP) slightly reduced this cell proliferation rate. For MC3T3-E1 cells, the addition of four cytokines examined (vitamin C, vitamin D, TGF-beta and BMP) did not significantly increase the cell proliferation while Dex with vitamin C and beta-GP slightly enlarged the cell proliferation. It can be pointed out that vitamin C in DME medium is indispensable for rapid proliferation of bone marrow stromal cells that contain many osteo-progenitor cells but is not effective for quick increase of osteoblastic MC3T3-E1 cells. This finding appears to contribute to tissue engineering therapy to fix bone and periodontal defects in dentistry.     

牙胚细胞与骨形态发生蛋白4转染的骨髓间充质干细胞相互诱导的体外实验研究

目的:探讨牙胚细胞和骨形态发生蛋白4(BMP4)转染的骨髓间充质干细胞(BMSCs)相互间的成牙诱导作用。方法:构建骨形态发生蛋白4慢病毒载体,转染SD大鼠骨髓间充质干细胞,并将骨髓间充质干细胞和BMP4转染的骨髓间充质干细胞分别与SD大鼠牙胚细胞按1∶1比例混合培养,同时将细胞分为5组,BMSCs组、BMP4/ BMSCs组、牙胚细胞组、BMSCs/牙胚细胞组(混合组1)、BMP4/ BMSCs/牙胚细胞组(混合组2),分别采用实时荧光定量PCR和western-blotting检测五组细胞Ⅰ型胶原蛋白、成釉蛋白、牙本质基质蛋白1、同源异型盒基因1成牙相关基因mRNA水平和蛋白水平相对表达量的变化。结果:与混合组1相比,混合组2Ⅰ型胶原蛋白、成釉蛋白、牙本质基质蛋白1、同源异型盒基因1 mRNA水平和蛋白水平表达量增多,差异有统计学意义(P<0.05)。结论:牙胚细胞与BMP4转染的骨髓间充质干细胞共培养,促进了成牙相关基因的表达,可作为组织工程牙的备选种子细胞。     

Potential role of PC-1 expression and pyrophosphate elaboration in the molecular etiology of the FGFR-associated craniosynostosis syndromes

BACKGROUND: Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is associated with the aberrant mineralization phenotype of the craniosynostosis syndromes. One critical aspect of mineralization involves the elaboration and transport of pyrophosphate into the extracellular matrix with subsequent enzymatic hydrolysis into phosphate. We have previously shown that FGF2 up-regulates expression of the pyrophosphate generating enzyme, PC-1, and the pyrophosphate channel, ANK, while down-regulating expression of the pyrophosphate hydrolyzing enzyme, tissue non-specific alkaline phosphatase in pre-osteoblastic, MC3T3E1(C4) cells. These results suggest that FGF/FGFR signaling may affect mineralization via changes in the elaboration and metabolism of pyrophosphate. OBJECTIVES: We are currently conducting experiments towards a more systematic analysis of PC-1 expression in osteoblastic cells, in order to more clearly elucidate the significance of pyrophosphate elaboration in the process of normal bone mineralization and in the molecular etiology of the FGFR-associated craniosynostosis syndromes. DESIGN: Towards this goal we have constructed a PC-1 gene promoter/firefly luciferase reporter construct, in order to more directly investigate the regulation of PC-1 by FGF/FGFR signaling in osteoblastic and non-osteoblastic cells. RESULTS AND CONCLUSIONS: Preliminary results confirm that FGF/FGFR signaling, either via treatment with FGF2 or via expression of a Crouzon syndrome-associated mutant FGFR2, induces PC-1 promoter activity in osteoblastic cells in culture. This appears to be a cell type specific phenomenon. These results suggest that the expression of PC-1 downstream of FGF signaling is an integral aspect of osteoblastic function, and that pyrophosphate elaboration may play a significant role in the pathology of craniosynostosis.     

Wnt和Notch通路在老龄个体骨髓间充质干细胞成骨中的调控

社会老龄化的加剧使老年个体骨损伤修复问题愈发突出,骨髓间充质干细胞(BMSC)是一种与骨代谢再生密切相关的骨髓细胞,其生物学特性(形态、表面特征、细胞周期、端粒酶以及细胞内活性氧簇水平等)以及增殖分化能力在生物体年龄影响下均发生了改变,成骨能力下降,影响了骨损伤的修复速度和质量.探索其中分子机制对改善老龄个体骨损伤康复有至关重要作用.参与调控的信号中,Wnt和Notch近年日益受到关注,二者对老龄BMSC成骨的调控有交互作用.老龄机体的氧化应激反应增加而生长因子生成减少,Wnt通路的转录因子β-catenin与叉头家族转录因子的亲和力增加,不再与T细胞因子和淋巴增强因子结合,故BMSC成骨减弱.同时老龄个体骨髓中BMSC数量减少,Notch抑制BMSC成骨来维持祖细胞池中BMSC的数量.Wnt和Notch之间还存在相互作用,如Notch过表达能够削弱Wnt的影响等.此外,BMP-Smad转录因子活性下降,Hedgehog信号通路下调,亦影响着BMSC的成骨分化.本文对老龄个体BMSC生物学性能变化及其成骨分化过程中信号通路的调控作用进行综述,为老龄个体骨相关性疾病的治疗提供新思路.     

The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis

ObjectiveThe bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix.DesignWe generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts.ResultsThe first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype.ConclusionsThese data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth.     

BMP-2、bFGF基因转染的大鼠骨髓干细胞与牙胚细胞在体外混合培养对成牙基因的影响

目的:观察骨形态发生蛋白-2和(或)碱性成纤维细胞生长因子转染的大鼠骨髓干细胞(BMSCs)和牙胚细胞在体外混合培养对牙本质基质蛋白1、成釉蛋白、Ⅰ型胶原蛋白、同源异型盒基因1的影响。方法:采用实时定量PCR和Western blot的方法,检测 BMP-2和(或)bFGF对成牙基因的影响。结果:对照组、bFGF组、BMP-2组、bFGF/BMP-2组四组间,AMBN、COLLAGEN-Ⅰ、DLX1、DMP1基因在mRNA和蛋白水平表达量上存在明显差异(P<0.05)。结论:BMP2和bFGF有一定的协同交互作用,BMP2/bFGF/BMSCs/牙胚细胞可以作为构建组织工程牙的种子细胞。     

In search of the ideal bone morphogenetic protein delivery system: in vitro studies on demineralized bone matrix,purified, and recombinant bone morphogenetic protein

The clinical use of recombinant bone morphogenetic protein (rBMP) is limited by the lack of a suitable delivery system. The bone morphogenetic protein (BMP) delivery system provided by nature is highly effective, and by studying purified BMP (BMP/NCP) and demineralized bone matrix (DBM), it may be possible to learn how to emulate nature's success. The current study used an in vitro muscle cell model to study the activity of BMP/NCP and DBM and the effects of extracellular matrix on BMP activity. C2C12 cells transiently exposed to recombinant human BMP-4 (rhBMP-4) rapidly increased their alkaline phosphatase (AP) activity to day 5, after which it steadily declined. Cells exposed to BMP/NCP or DBM continued to increase their AP activity over the 14-day culture. If BMP/NCP was treated to remove a 22-kd protein, it became water-soluble and exhibited a similar activity pattern to rhBMP-4. Cells cultured on collagen type I, fibronectin, and hyaluronic-coated surfaces demonstrated increased AP activity when exposed to rhBMP-4 or BMP/NCP compared with cells cultured on bovine serum albumin or poly-l-lysine. These results suggest that the natural BMP delivery system operates both by binding to the BMP molecule and slowly releasing it into the extracellular milieu and by interacting with the responding cells through cell-matrix receptors to enhance the cellular response to BMP.     

The signaling pathways involved in non-coding RNA regulation during osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis

Periodontitis is a prevalent oral disease caused by chronic inflammation of the periodontal tissues surrounding the teeth, which can lead to bone loss, tooth loosening, and even tooth loss. This inflammation has a negative impact on the osteogenic differentiation capacity of periodontal tissue-derived cells. Non-coding RNAs (ncRNAs) are a class of RNA molecules that do not encode proteins but can regulate various physiological processes. In this review, we summarized the critical signaling pathways that ncRNAs modulate in osteogenic differentiation of periodontal tissue-derived cells, such as the Wnt, BMP/Smad, NF-κB, and PI3-K/Akt/mTOR pathways. This comprehensive exploration of ncRNA-mediated modulation offers fresh and promising insights for prospective approaches in the management of periodontitis and the advancement of periodontal regeneration therapies.     

Expression of bone morphogenetic proteins in normal human intramembranous and endochondral bones

Bone morphogenetic proteins (BMPs) are growth and differentiation factors that have been purified and widely accepted to be the most important regulators in the processes of bone formation. The aim of this study was to identify the BMPs that are expressed in normal human bone, and to investigate the specific pattern of BMP2-BMP9 expression in normal human intramembranous and endochondral bone to maintain homeostasis, as well as in ex vivo primary cell culture of human osteoblasts from intramembranous and endochondral bone. Semi-quantitative RT-PCR indicated that 2 types of bone of different embryological origin have distinct patterns of BMP expression. BMP3, 4, 7 and 8 were strongly expressed in normal intramembranous bone compared to endochondral bone, whereas BMP2 and 5 were highly expressed in endochondral bone. The expression of BMP9 and BMP15 in human bone was identified for the first time. From the very similar expression patterns of BMPs in fresh normal bone and ex vivo osteoblastic cell culture, it can be proposed that the different proportions of BMPs in normal human intramembranous and endochondral bone needed to maintain normal homeostasis.     

Regulation of osteoclast function via Rho-Pkn3-c-Src pathways

BackgroundWnt signaling pathways are largely divided into the β-catenin-dependent canonical pathway and β-catenin-independent non-canonical pathways. The roles of Wnt signaling in bone metabolism have been extensively investigated.We previously attempted to clarify the roles of Wnt-non-canonical signaling in bone resorption and demonstrated that Wnt5a-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling promoted osteoclast differentiation by enhancing RANK expression in osteoclast precursor cells. However, the roles of Wnt5a-Ror2 signaling in osteoclast function remain unclear.HighlightTrabecular bone mass was significantly greater in osteoclast-specific Ror2-deficient (Ror2^ΔOCL/ΔOCL) mice than in control mice due to the decreased bone-resorbing activity of osteoclasts. Wnt5a-Ror2 signaling activated Rho in osteoclasts via dishevelled-associated activator of morphogenesis 2 (Daam2). The expression of protein kinase N3 (Pkn3), a Rho effector, increased during osteoclast differentiation. Trabecular bone mass was significantly greater in Pkn3-deficient mice than in wild-type mice due to the decreased bone-resorbing activity of osteoclasts. Pkn3 bound to c-Src and Pyk2 in a Wnt5a-Ror2 signaling-dependent manner, thereby enhancing the kinase activity of c-Src in osteoclasts. The binding of Pkn3 to c-Src was essential for the bone-resorbing activity of osteoclasts.ConclusionWnt5a-Ror2 signaling promotes the bone-resorbing activity of osteoclasts by activating the Daam2-Rho-Pkn3-c-Src pathways. Pkn3 inhibitors, therefore, have potential as therapeutic agents for osteoporosis and bone destruction in inflammatory diseases.