The role of Osterix protein in the pathogenesis of peripheral ossifying fibroma

Abstract Peripheral ossifying fibroma (POF) is a reactive lesion of oral tissues, associated with local factors such as trauma or presence of dental biofilm. POF treatment consists of curettage of the lesion combined with root scaling of adjacent teeth and/or removal of other sources of irritants. This study aimed to analyze the clinical and pathological features of POF and to investigate the immunoexpression of Osterix and STRO-1 proteins. Data such as age, gender, and size were obtained from 30 cases of POF. Microscopic features were assessed by conventional light microscopy using hematoxylin-eosin staining and immunohistochemical markers, and by polarized light microscopy using Picrosirius red staining. The age range was 11-70 years and 70% of the patients were female. Moreover, the size of POF varied from 0.2 to 5.0 cm; in 43.33% of the cases, the mineralized content consisted exclusively of bony trabeculae. The immunohistochemical analysis showed nuclear staining for Osterix in 63% and for STRO-1 in 20% of the cases. Mature collagen fibers were observed in mineralized tissue in 76.67% of the cases. The clinical and microscopic features observed were in agreement with those described in the literature. Osterix was overexpressed, while STRO-1 was poorly expressed. Osterix was expressed particularly in cells entrapped in and around mineralized tissue, indicating the presence of a stimulus that triggers the differentiation of these cells into osteoblasts or cementoblasts, i.e., cells that produce mineralized tissue. Based on our results, Osterix may play a role in the pathogenesis of POF.

Effect of Total Ravonoids of Herba Epimedium on BMP-2/RunX2/OSX Signaling Pathway during Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).</p><p><b>METHODS</b>Passage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR.</p><p><b>RESULTS</b>Compared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group.</p><p><b>CONCLUSION</b>20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.</p>

Expression of osterix gene in the early stage of cranio-maxillofacial development in zebrafish / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression patterns of osterix in the early stage of cranio-maxillofacial developmental in zebrafish and to prepare for a further research of osterix gene in bone and tooth development.</p><p><b>METHODS</b>The osterix templates were amplified by PCR to generate DIG labeled antisense and sense probes. Whole mount in situ hybridization was used to analyze the expression patterns of osterix in the early stage cranio-maxillofacial development of zebrafish. The expression patterns of osterix gene in mineralization progresses of cranial and maxillofacial bones were compared. The osterix gene expression in tooth development and mineralization was highlighted by alizarin red staining.</p><p><b>RESULTS</b>Specific DIG labeled probes of osterixwere synthesized successfully. The whole mount in situ hybridization showed that the osterix expression was in the intramembranous ossification at 3 days post fertilization(dpf) and 4 dpf. The specific osterix expression in tooth at 5 dpf and 6 dpf were also observed. The sense probe served as a negative control. Osterix expressed in the unmineralized early bone matrix, the tooth matrix of the primary tooth(3V(1), 5V(1)) and the first replacement tooth(4V(2)).</p><p><b>CONCLUSIONS</b>Our findings showed that osterix might play roles in the process of the early mineralized bone matrix changing into the late mature mineralized bone matrix and the process of development and mineralization of tooth crown matrix.</p>

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

BACKGROUND: Tridaxprocumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Hedgehog signaling pathway and osteoporosis / 中国骨伤

Hedgehog signaling pathway is a conserved and important signaling pathway involved in proliferation and differentiation of many types of cells. Latest studies have found that Hedgehog signaling pathway may induce MSCs osteoblast differentiation by increasing the expression of the Runx2 and Osx and inhibit MSCs differentiate to adipocyte. Hedgehog signaling pathway may also promote osteoblast proliferation by regulating cyclin. This review summarizes the mechanism that Hedgehog signaling pathway regulates osteoblast differentiation and proliferation,and concludes that Hedgehog signaling pathway can regulate bone metabolism. It might provide new ideas for the treatment of osteoporosis.

Bone morphogenetic protein-2 gene controls tooth root development in coordination with formation of the periodontium / 国际口腔科学杂志·英文版

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.

KDM6B epigenetically regulates odontogenic differentiation of dental mesenchymal stem cells / 国际口腔科学杂志·英文版

Mesenchymal stem cells (MSCs) have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B (also known as JMJD3) was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate (ALP) activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 (osterix, OSX), as well as extracellular matrix genes BGLAP (osteoclacin, OCN) and SPP1 (osteopontin, OPN), was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 (BMP2) promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.

Calcium hydroxide regulates bone sialoprotein gene transcription in human dental pulp cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To analyze the effects of calcium hydroxide [Ca(OH)2] on transcription of the bone sialoprotein (BSP) gene in human dental pulp cells.</p><p><b>METHODS</b>Human dental pulp tissues were collected from extracted teeth for orthodontic reason. In cell culture media, different dose (0.012, 0.120, 0.400 and 1.200 mmol/L) of Ca(OH)2 was added. Total RNA of cells were extracted. The best dose of Ca(OH)2 on human BSP was determined with the real-time polymerase chain reaction (PCR). Further, the time (0, 3, 6, 12, 24 h) effects of the best dose Ca(OH)2 on human BSP, runt-related transcription factor-2 (Runx-2) and osterix (OSX) mRNA levels were determined with PCR. Further method included transient transfection assays, linking chimeric constructs of the human BSP gene promoter to a luciferase reporter gene, then ransfected using lipofectamine in cells and measured the luciferase activities of BSP gene promoter.</p><p><b>RESULTS</b>With the real-time PCR, the optimal Ca(OH)2 concentration was determined as 1.200 mmol/L. With this concentration at different time points (0, 3, 6, 12 and 24 h), the levels of BSP mRNA increased at 6 h (1.45 ± 0.36), reached maximal at 12 h (2.66 ± 0.18); the levels of Runx-2mRNA increased at 6 h (2.38 ± 0.08), at 12 h (2.73 ± 0.16), and decreased at 24 h. OSX mRNA could be recognized at 12 h, reached maximal levels at 24 h (3.30 ± 0.062). Transient transfection assays showed that treatment of human dental pulp cells with Ca(OH)2 (1.200 mmol/L) increased the luciferase activities of the constructs between -84LUC and -868LUC at 12 h (2.00 ~ 2.60 fold).</p><p><b>CONCLUSIONS</b>This study demonstrate that Ca(OH)2 could stimulate BSP transcription between -84LUC and -868LUC in the human BSP gene promoter in human dental pulp cells.</p>

Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of mycophenolic acid (MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>MSCs were treated with MPA at the concentration of 1 μ mol/L, 10 μ mol/L, 50 μ mol/L, and 100 μ mol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5'-monophosphate dehydrogenase (IMPDH) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining, Ca(2+) quantification and real-time PCR.</p><p><b>RESULTS</b>In the range of 1 μ mol/L to 100 μ mol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH I and IMPDH II. von Kossa staining and Ca(2+) quantification indicated that MPA inhibited osteogenic differentiation of MSCs, and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix.</p><p><b>CONCLUSION</b>MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration-and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.</p>

Pericyst may be a new pharmacological and therapeutic target for hydatid disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most hydatid cysts with calcified walls are biologically and clinically silent and inactive. Transforming growth factor-beta 1 (TGF-β1) plays a critical role in the calcification process of cells. The aim of this study was to assess the effect of modulating TGF-β1 signaling on the calcification of hydatid cysts.</p><p><b>METHODS</b>Pericyst cells isolated from hepatic hydatid cysts were cultured with osteogenic media. These cells were assessed for alkaline phosphatase activity and mineralization capacity using Alizarin Red staining. Cells were also treated with recombinant human TGF-β1 and TGF-β inhibitor, and the expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) were analyzed using Western blotting. The effects of inhibiting TGF-β1 signaling on calcification of pericyst walls were assessed using different doses of TGF-β inhibitor for 7 weeks in a preclinical disease model of liver cystic echinococcosis.</p><p><b>RESULTS</b>Cells within the pericyst displayed high levels of alkaline phosphatase activity and mineralized nodule formation, as induced by osteogenic media. These activities, as well as expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) could be inhibited by addition of recombinant human TGF-β1 (rhTGF-β1) and enhanced by TGF-β inhibitor. In the animal model of cystic echinococcosis, inhibition of TGF-β1 signaling increased calcification of the pericyst wall, which was associated with decreased cyst load index and lower viability of protoscoleces.</p><p><b>CONCLUSIONS</b>Cells within the pericysts adopt an osteoblast-like phenotype and have osteogenic potential. Inhibition of TGF-β1 signaling increases hydatid cyst calcification. Pharmacological modulation of calcification in pericysts may be a new therapeutic target in the treatment of hydatid disease.</p>

Effect of mechanical strain on differentiation of mesenchymal stem cells into osteoblasts / 生物医学工程学杂志

This study sought to elucidate the effect of mechanical strain on the differentiation of mesenchymal stem cells into osteoblasts. Under the conditons of inducing osteoblasts, Immunohistochemical methods and RT-PCR technology were applied in osteogenic supplements medium to detect: (1) the expression of Alkaline phosphatase (ALP), Type I collagen (COL I ), Osterx (Osx) and Osteocalcin (OCN) mRNA, with cyclic strain (3%, 0.5 Hz) applied for 15 min, 30 min, 1 h, 2 h, 4 h, 3 d, 7 d, 14 d; (2) the expression of Osx mRNA and OCN mRNA with 3% strain for 1 h. The results showed: (1) ALP mRNA expression was higher at 7 days; COL I mRNA expression was greater obviously at 7 days and 14 days than that at 3 days and that of the unstrained cells; (2) the expression of Osx mRNA was up-regulated after 15min by strain stimulation,which was significantly increased at 30 min and 1 h in the unstrained cells. The expression of OCN mRNA was not affected in the unstrained cells at 15 min, whereas strain could promote the expression of OCN mRNA at this period. The expression of OCN mRNA was more obviously upregulated in the strained cells at 30 min and 1 h when compared with that in the unstrained cells; (3) the strain (1% and 3%) significantly promoted the expression of Osx mRNA; 10% strain had a little effect on Osx mRNA expression. The expression of OCN mRNA was up-regulated by 3% strain, whereas it had little effect at 1% and 10% strain. In summary, mechanical strain can promote the differentiation of mesenchymal stem cells into osteoblasts.