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Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by β-glycerophosphate (β-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in β-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In β-GP-treated VSMCs, BK channel opener NS1619 (20 μM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 μM) caused the opposite effects. In primary cultured VSMCs from BK -/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK -/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg -1  ·d -1 ) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 μM) or FoxO1 inhibitor AS1842856 (3 μM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Background: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches., Methods: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo., Results: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment., Conclusion: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Objective: To investigate the therapeutic effects of PERK activator CCT020312 (CCT) on inflammation-mediated osteoporosis (IMO) in ovariectomized rats., Methods: Rats were divided into Sham, IMO, IMO + 1 mg/kg CCT and IMO + 2 mg/kg CCT groups. IMO models were constructed by bilateral ovariectomy (OVX) on 1st day followed by injection with magnesium silicate (Talc) on the 59th day. Sham rats did not undergo OVX surgery and were injected with saline instead of Talc. From 60th to 79th day, rats were treated with DMSO (vehicle control) in the Sham and IMO groups, and 1 or 2 mg/kg CCT020312 in treatment groups. Osteopontin (OPN), osteocalcin (OCN), tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type I collagen (CTX-I), and pro-inflammatory factors were measured on the 80th day. ProdigyDEXA was used to evaluate bone mineral density and content (BMD/BMC). Bone volume/total volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N), and trabecular separation (Tb.Sp) was assessed using 3D micro-CT scanner., Results: CCT up-regulated Conn.D, BV/TV, and Tb.N, but down-regulated Tb.Sp in IMO rats. Besides, the declined femoral BMD and BMC in IMO rats were elevated after CCT treatment. Besides, IMO rats represented declined OPN and OCN, as well as increased TRAP, CTX-I, and pro-inflammatory factors, whereas those in the treatment groups were ameliorated regarding these indexes, with 2 mg/kg CCT showing better effect., Conclusion: PERK activator CCT020312 can be served as a new therapeutic option for the protection against bone loss in the OVX rat model associated with inflammation probably by manipulating inflammatory factors.

Background: Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathway is associated with the pathogenesis of pulmonary hypertension (PH). However, the effect of icotinib, a first generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), on PH remains to be elucidated., Methods: PH rat model was established by a single intraperitoneal injection of monocrotaline (MCT, 60 mg/kg). Icotinib (15, 30, and 60 mg/kg/day) was administered by oral gavage from the day of MCT injection. After 4 weeks, hemodynamic parameters and histological changes of the pulmonary arterial vessels were assessed, and the phenotypic switching of pulmonary arterial smooth muscle cells (PASMCs) was determined in vivo. Moreover, the effects of icotinib (10 µM) on epidermal growth factor (EGF, 50 ng/ml)-stimulated proliferation, migration, and phenotypic switching of human PASMCs were explored in vitro., Results: Icotinib significantly reduced the right ventricular systolic pressure and right ventricle hypertrophy index in rats with MCT-induced PH. Moreover, icotinib improved MCT-induced pulmonary vascular remodeling. The expression of contractile marker (smooth muscle 22 alpha (SM22α)) and synthetic markers (osteopontin (OPN) and vimentin) in pulmonary artery was restored by icotinib treatment. In vitro, icotinib suppressed EGF-induced PASMCs proliferation and migration. Meanwhile, icotinib inhibited EGF-induced downregulation of α-smooth muscle actin and SM22α and upregulation of OPN and Collagen I in PASMCs, suggesting that icotinib could inhibit EGF-induced phenotypic switching of PASMCs. Mechanistically, these effects of icotinib were associated with the inhibition of EGFR-Akt/ERK signaling pathway., Conclusions: Icotinib can attenuate MCT-induced pulmonary vascular remodeling and improve PH. This effect of icotinib might be attributed to preventing PASMC dysfunction by inhibiting EGFR-Akt/ERK signaling pathway., (© American Journal of Hypertension, Ltd 2020. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Central to the development of diabetic macro- and microvascular disease is endothelial dysfunction, which appears well before any clinical sign but, importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates nuclear factor of activated T cells (NFAT) in conduit and medium-sized resistance arteries and that NFAT blockade abolishes diabetes-driven aggravation of atherosclerosis. In this study, we test whether NFAT plays a role in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita ( Ins2 +/- ) mice when compared with nondiabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin microvessels, resulting in improved microvascular function, as assessed by laser Doppler imaging and iontophoresis of acetylcholine and localized heating. This improvement was abolished by pretreatment with the nitric oxide (NO) synthase inhibitor l- N G -nitro-l-arginine methyl ester, while iontophoresis of the NO donor sodium nitroprusside eliminated the observed differences. A-285222 treatment enhanced dermis endothelial NO synthase expression and plasma NO levels of diabetic mice. It also prevented induction of inflammatory cytokines interleukin-6 and osteopontin, lowered plasma endothelin-1 and blood pressure, and improved mouse survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes., (© 2019 by the American Diabetes Association.)

Osteoporosis is a metabolic disease characterized by reduced osteoblast differentiation and proliferation. Oxidative stress plays a role in the pathogenesis of osteoporosis. Aucubin (AU), an iridoid glycoside, was previously shown to promote osteoblast differentiation. We investigated the effects of AU on MG63 human osteoblast-like cells treated with dexamethasone (Dex) or hydrogen peroxide (H 2 O 2 ) to induce oxidative damage. AU protected MG63 cells against apoptosis, and promoted increased expression of cytokines associated with osteoblast differentiation, including collagen I, osteocalcin (OCN), osteopontin (OPN), and osterix. In Dex- and H 2 O 2 -treated MG63 cells, AU also enhanced the expression of anti-oxidative stress-associated factors in the nuclear respiratory factor 2 signaling pathway, including superoxide dismutases 1 and 2, heme oxygenases 1 and 2, and catalase. In vivo , using a Dex-induced mouse model of osteoporosis, AU promoted increased cortical bone thickness, increased bone density, and tighter trabecular bone. Additionally, it stimulated an increase in the expression of collagen I, OCN, OPN, osterix, and phosphorylated Akt and Smads in bone tissue. Finally, AU stimulated the expression of cytokines associated with osteoblast differentiation in bone tissue and serum. Our data indicate AU may have therapeutic efficacy in osteoporosis.

Multiple lines of evidence suggest a link between depression and osteoporosis in elderly people. Receptor activator of nuclear factor-κB ligand (RANKL) plays a role in the pathology of osteoporosis, and anti-RANKL antibody has been used in the treatment of osteoporosis. In this study, we investigated whether anti-mouse RANKL antibody could attenuate depression-like phenotypes, inflammatory bone markers and bone mineral density (BMD) in male susceptible mice after chronic social defeat stress (CSDS). We measured plasma levels of inflammatory bone markers, including osteoprotegerin (OPG), RANKL, and osteopontin. A single intravenous injection of anti-RANKL (2 mg/kg) elicited rapid antidepressant effects in CSDS susceptible mice. Furthermore, anti-RANKL significantly improved the increased plasma levels of RANKL and decreased OPG/RANKL ratio in CSDS susceptible mice. Moreover, anti-RANKL significantly attenuated the decreased BMD in CSDS susceptible mice. Interestingly, there is a positive correlation between anhedonia-like behavior and OPG/RANKL ratio in mice. These findings demonstrate that anti-RANKL may have beneficial effects in depression-like phenotype and abnormalities in bone functions of CSDS susceptible mice. It is, therefore, likely that anti-human RANKL antibody (i.e., Denosumab) would be a potential therapeutic drug for depression and osteoporosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To evaluate and compare plasma osteopontin (OPN, a candidate prostate cancer biomarker) levels in prostate cancer patients receiving radiotherapy or combined radiotherapy or hormone therapy., Methods: OPN levels were determined by ELISA in 40 prostate cancer patients eligible for radiotherapy (n=18 radiotherapy alone, n=22 combined radiotherapy and hormone therapy) before the start of irradiation, during treatment, and one month after its completion., Results: OPN levels were significantly higher (p=0.02) in prostate cancer patients after receiving radiotherapy compared to baseline. In a subgroup analysis, there were no differences in OPN levels before and after treatment in patients undergoing radiotherapy alone, but OPN levels were significantly higher in patients after radiotherapy with hormone therapy compared to baseline (p=0.04) and in patients during radiotherapy compared to baseline (p=0.03)., Conclusions: Radiotherapy can increase plasma OPN concentrations in patients with prostate cancer, and radiotherapy may interact with hormone therapy to increase OPN concentrations. These differences suggest that OPN is worthy of further study as a predictive biomarker.

Background: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma., Methods: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene., Results: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1., Conclusion: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.

BACKGROUND Renal stones are the accumulated or deposited crystals that form and appear in supersaturated urine. This study aimed to the investigate the therapeutic effects of Huashi Pill on clearance of renal stones. MATERIAL AND METHODS Sprague Dawley rats were divided into normal control, positive control, low-dosage Huashi Pill, medium-dosage Huashi Pill, and high-dosage Huashi Pill groups. A renal rat model was established by using ethylene glycol, ammonium chloride, and calcium gluconate. The urinary pH, urine protein, and uric acid levels, as well as the calcium, magnesium, and phosphorus levels were examined. The blood urea nitrogen (BUN) and creatinine (Cr) levels were also evaluated. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) levels were evaluated. Crystal formation and calcium deposits were examined using hematoxylin and eosin (H and E) staining and von Kossa staining, respectively. Osteopontin (OPN) expression was evaluated with quantitative real-time polymerase chain reaction assay and immunohistochemical assay. RESULTS A renal stone rat model was successfully established. Huashi Pill significantly improved water and food intake and enhanced pH value of urine (P<0.05). Huashi Pill significantly improved the liver functions by decreasing ALT and TBIL levels (P<0.05). Huashi Pill regulated the amounts of microelements. Huashi Pill significantly decreased the urine protein, uric acid, and Cr levels (P<0.05). Huashi Pill inhibited formation of stone crystals and reduced the insoluble calcium deposition. Huashi Pill significantly downregulated expression of OPN in the kidney tissues of renal rat models (P<0.05). CONCLUSIONS Huashi Pill inhibited stone formation by regulating urine biochemical indexes and reducing OPN expression in kidney tissue in a renal stone rat model.

Background We have developed a peptide vaccine named ATRQβ-001, which was proved to retard signal transduction initiated by angiotensin II (Ang II). Ang II was implicated in abdominal aortic aneurysm (AAA) progression, but whether the ATRQβ-001 vaccine would prevent AAA is unknown. Methods and Results Ang II-infused ApoE -/- mice and calcium phosphate-induced AAA in C57BL/6 mice were used to verify the efficiency of ATRQβ-001 vaccine in AAA. Results demonstrated that the vaccine effectively restrained the aneurysmal dilation and vascular wall destruction of aorta in both animal models, beyond anti-hypertensive effects. In Ang II-induced AAA vascular sections, Immunohistochemical staining showed that the vaccine notably constrained vascular inflammation and vascular smooth muscle cell (VSMC) phenotypic transition, concurrently reduced macrophages infiltration. In cultured VSMC, the anti-ATR-001 antibody inhibited osteopontin secretion induced by Ang II, thereby impeded macrophage migration while co-culture. Furthermore, metalloproteinases and other matrix proteolytic enzymes were also found to be limited by the vaccine in vivo and in vitro. Conclusions ATRQβ-001 vaccine prevented AAA initiation and progression in both Ang II and calcium phosphate-induced AAA models. And the beneficial effects were played beyond decrease of blood pressure, which provided a novel and promising method to take precautions against AAA.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures., Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells., Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR., Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation., Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

Objective: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo., Methods: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs., Results: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 μm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups., Conclusion: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

Objective: The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin)., Material and Methods: A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level., Results: Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days., Conclusion: There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats.

Chronic inhibition of nitric oxide synthase by N(ω)-nitro-L-arginine methyl ester (L-NAME) causes progressive renal injury and systemic hypertension. Angiotensin II (Ang II) has been conventionally regarded as one of the primary causes of renal injury. We reported previously that such renal injury was almost completely suppressed by both an Ang II type I receptor blocker and an aldosterone antagonist. The aldosterone antagonist also inhibited the systemic Ang II elevation. Therefore, it remains to be elucidated whether Ang II or aldosterone directly affects the development of such renal injury. In the present study, we investigated the role of aldosterone in the pathogenesis of renal injury induced by L-NAME-mediated chronic nitric oxide synthase inhibition in male Wistar rats (aged 10 wk). Serial analyses demonstrated that the renal injury and inflammation in L-NAME-treated rats was associated with elevation of both Ang II and aldosterone. To investigate the direct effect of aldosterone on the renal injury, we conducted adrenalectomy (ADX) and aldosterone supplementation in L-NAME-treated rats. In ADX rats, aldosterone was undetectable, and renal injury and inflammation were almost completely prevented by ADX, although systemic and local Ang II and blood pressure were still elevated. Aldosterone supplementation reversed the beneficial effect of ADX. The present study indicates that aldosterone rather than Ang II plays a central and direct role in the pathogenesis of renal injury by L-NAME through inflammation, independent of its systemic hemodynamic effects.

Background and Objective: The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells., Material and Methods: A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively., Results: Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of β-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells., Conclusion: Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of β-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: This study investigated the influence of fluoride levels on the temporal synthesis of bone-associated glycoproteins, which have been assigned prominent roles in regulating crystal growth, size and shape during the mineralization process., Materials and Methods: Bone marrow stromal cells were isolated from male Wistar rats and cultured under mineralizing conditions, supplemented with 0 M, 10(-7) M or 10(-5) M sodium fluoride. The presence of bone-associated glycoproteins was examined 2-13 days post-reseeding by immunocytochemical localization. Results: All bone-associated glycoproteins increased in 10(-7) M fluoride, compared to untreated controls, particularly at days 6 and 13 in culture. Conversely, higher 10(-5) M fluoride concentrations decreased glycoprotein levels, compared to controls., Conclusions: Results highlight a differential effect of fluoride concentration on glycoprotein synthesis by osteoblasts.

Introduction: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries., Methods: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction., Results: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7., Conclusions: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: OrthoMTA (BioMTA, Seoul, Korea) and Endocem MTA (Maruchi, Wonju-si, Korea) were recently developed to overcome the disadvantages of ProRoot MTA (Dentsply, Tulsa, OK). This study aimed to compare the biological properties of OrthoMTA and Endocem MTA with those of ProRoot MTA using the preosteoblastlike cell line MC3T3-E1., Methods: The setting times of calcium silicate-based cements (CSCs) and their effects on the pH of distilled water during storage were determined according to ISO standards. MC3T3-E1 cells were cultured with ProRoot MTA, OrthoMTA, and Endocem MTA. The viability of the cells was assessed using the Cell Counting Kit-8 assay (Dojindo Laboratory, Kumamoto, Japan) on the supernatants of CSCs, and the cells' osteopontin production was determined by an enzyme-linked immunosorbent assay on a culture with the materials on days 3 and 7 of incubation., Results: Endocem MTA exhibited a significantly shorter setting time (15.3 ± 0.5 minutes) than did ProRoot MTA and OrthoMTA (318.0 ± 56.0 and 324.3 ± 2.1 minutes, P < .05). Additionally, all CSCs caused their storage water to become highly alkaline after 7 days. OrthoMTA was significantly more cytotoxic than ProRoot and Endocem MTA (P < .05). ProRoot MTA induced significantly more OPN production than OrthoMTA and Endocem MTA on both days 3 and 7 (P < .05)., Conclusions: ProRoot MTA appeared to be superior to OrthoMTA and Endocem MTA in terms of biological properties although Endocem MTA exhibited the shortest setting time and presented lower cytotoxicity with osteoblastlike cells., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the effect of astaxanthin on renal angiotensin-I converting enzyme (ACE) levels, osteopontin (OPN) and transforming growth factor β1 (TGF-β1) expressions and the extent of crystal deposition in experimentally induced calcium oxalate kidney stone disease in a male Wistar rat model. To compare the efficacy of astaxanthin treatment with a currently used treatment strategy (citrate administration) for kidney stones., Materials and Methods: The expression of OPN was assessed by immunohistochemistry. One step reverse transcriptase polymerase chain reaction followed by densitometry was used to assess renal OPN and TGF-β1 levels. Renal ACE levels were quantified by an enzyme-linked immunosorbent assay method. Crystal deposition in kidney was analysed by scanning electron microscopic (SEM)-energy-dispersive X-ray (EDX)., Results: The renal ACE levels and the expression of OPN and TGF-β1 were upregulated in the nephrolithiasis-induced rats. Astaxanthin treatment reduced renal ACE levels and the expression OPN and TGF-β1. SEM-EDX analysis showed that crystal deposition was reduced in the astaxanthin-treated nephrolithiatic group. Astaxanthin treatment was more effective than citrate administration in the regulation of renal ACE levels, OPN and TGF-β1 expressions., Conclusions: Astaxanthin administration reduced renal calcium oxalate crystal deposition possibly by modulating the renal renin-angiotensin system (RAS), which reduced the expression of OPN and TGF-β1 levels. Astaxanthin administration was more effective than citrate treatment in reducing crystal deposition and down-regulating the expression of OPN and TGF-β1., (© 2013 The Authors. BJU International © 2013 BJU International.)

Fibroblast growth factor 9 (FGF9) plays complicated and crucial roles in bone formation, and the biologic effect of FGF9 may depend on the gene dosage, developmental stage, cell type, or interactions with other cytokines. In this study, we demonstrated that FGF9 enhanced the phosphorylation of extracellular regulated protein kinases 1/2 in calvaria-derived mesenchymal cells. However, the inhibitory effect of FGF9 on the osteogenic differentiation of calvaria-derived mesenchymal cells did not depend on the phosphorylation of extracellular regulated protein kinases 1/2. Combined with the previous findings that FGF9 promotes dental pulp stem cells chondrogenesis in vitro, we suggest that FGF9 may be applied to promote chondrogenesis and inhibit osteogenesis in mesenchymal stem cells in vitro.

Introduction: The aim of this study was to compare the biocompatibility and odontogenic potential of newly developed Bioaggregate (BA) and Micromega MTA (MMTA) with ProRoot MTA (PMTA) and intermediate restorative material (IRM) by using human dental pulp cells., Methods: Biocompatibility was assessed by an 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. Differentiation was evaluated by alkaline phosphatase (ALP) activity, alizarin red staining, and reverse transcriptase-polymerase chain reaction for the maker genes. The levels of inflammatory mediators and cytokines were measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay., Results: PMTA, BA, and MMTA exhibited equally good biocompatibility, whereas IRM showed cytotoxicity compared with these materials. PMTA, BA, and MMTA increased the ALP activity, promoted mineralization nodule formation, and enhanced the mRNA expression level of the osteogenic/odontogenic markers (ALP, osteopontin, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1) compared with IRM. The levels of proinflammatory mediators and proinflammatory cytokines were lower in PMTA, BA, and MMTA compared with the IRM group., Conclusions: Collectively, the biocompatibility, odontogenic potentials, and inflammatory response of BA and MMTA are equal to those of PMTA and superior to those of IRM., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion., (Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Integrin-linked kinase (ILK) is a serine/threonine kinase that has been linked to human and experimental heart failure, but its role in the heart is not fully understood., Methods and Results: To define the role of cardiomyocyte ILK, we generated cardiac-specific ILK knockout mice using α-myosin heavy chain-driven Cre expression. Cardiac-specific ILK knockout mice spontaneously developed lethal dilated cardiomyopathy and heart failure with an early increase in apoptosis, fibrosis, and cardiac inflammation. To identify downstream effectors, we used deep sequence analysis of gene expression to compare comprehensive transcriptional profiles of cardiac-specific ILK knockout and wild-type hearts from 10-day-old mice before the development of cardiac dysfunction. Approximately 2×10(6) cDNA clones from each genotype were sequenced, corresponding to 33 274 independent transcripts. A total of 93 genes were altered, using nominal thresholds of >1.4-fold change and P<0.001. The most highly upregulated gene was osteopontin (47-fold increase; P=9.6×10(-45)), an inflammatory chemokine implicated in heart failure pathophysiology. ILK also regulated osteopontin expression in cardiomyocytes in vitro. Importantly, blocking antibodies to osteopontin mitigated but did not fully rescue the functional decline in cardiac-specific ILK knockout mice., Conclusions: Cardiomyocyte-specific ILK deletion leads to a lethal cardiomyopathy characterized by cardiomyocyte death, fibrosis, and inflammation. Comprehensive profiling identifies ILK-dependent transcriptional effects and implicates osteopontin as a contributor to these phenotypes.

This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors., (Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Objectives: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin., Method: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers., Results: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population., Conclusion: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 genes. The combined analysis of the suppressor of cytokine signaling 3 and IL28B genotypes more effectively predicted sustained virologic response than IL28B analysis alone.

Bone regeneration around titanium (Ti) implants is a relatively slow process. The c-myb transcription factor has been associated with high proliferation and differentiation rates in bone. This study analyzed whether c-myb can enhance new bone surrounding the implant. In vitro overexpressed chitosan-gold nanoparticles conjugated with plasmid DNA/c-myb (Ch-GNPs/c-myb)-coated Ti surfaces were associated with enhanced expression of the osteogenic molecules osteopontin (OPN), runt-related transcription factor 2 (RUNX-2), and bone morphogenetic proteins (BMP2/7) in MC-3T3E1 osteoblast cells. Further, to determine its in vivo effect, we inserted Ch-GNPs/c-myb-coated Ti implants into rat mandibles. One and 4 wks post-implantation, mandibles were examined by microcomputed tomography, immunohistochemistry, and hematoxylin & eosin staining. The microcomputed tomography analysis demonstrated that c-myb overexpression increased the density and volume of newly formed bone surrounding the implants, compared with those in controls (p < .05). Further, c-myb increased the number of cells expressing BMP2/7 and aided in the increase of new bone (p < .05). These results support the view that c-myb overexpression accelerates new bone surrounding implants and can serve as a potent molecule in promoting tissue regeneration around dental implants. The recipient rat used in this system provides an excellent in vivo model for studies of bone regeneration.

Introduction: The aim of this study was to investigate the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating tricalcium silicate (C3S)-driven proliferation and biomineralization of human dental pulp cells (hDPCs) in vitro., Methods: Human DPCs were cultured in C3S-containing medium and compared with untreated controls. Cell viability was measured by the methyl-thiazol-tetrazolium assay. Biomineralization was assessed by staining calcium deposits on the extracellular matrix with von Kossa and alizarin red S stains. Phosphorylated ERK1/2 was evaluated by immunoblotting. The ERK1/2 inhibitor U0126 was used to assess the role of this pathway on stage of the cell cycle and mineralization-dependent gene expressions of hDPCs by using flow cytometry and real-time polymerase chain reaction, respectively. Data were analyzed by analysis of variance followed by the Student-Newman-Keuls post hoc test, with significance set at P < .05., Results: The viability and biomineralization of hDPCs were promoted by C3S extracts (P < .05). Phosphorylated ERK1/2 strongly appeared after hDPCs were cultured in the C3S extracts for 30 minutes. Moreover, inhibition of the ERK1/2 pathway in C3S-treated hDPCs decreased proliferation and the expression of mineralization-dependent genes, including collagen type I, dentin sialophosphoprotein, osteopontin, and osteocalcin (P < .05)., Conclusions: C3S stimulated the proliferation and biomineralization of hDPCs in vitro, with the ERK1/2 pathway playing a key role in the regulation of these effects., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: To assess the effect of topical application of melatonin to the gingiva on salivary fluid concentrations of acid phosphatase, alkaline phosphatase, osteopontin, and osteocalcin., Study Design: Cross-sectional study of 30 patients with diabetes and periodontal disease and 30 healthy subjects. Diabetic patients were treated with topical application of melatonin (1% orabase cream formula) once daily for 20 days and controls with a placebo formulation., Results: Before treatment with melatonin, diabetic patients showed significantly higher mean salivary levels of alkaline and acid phosphatase, osteopontin and osteocalcin than healthy subjects (P < 0.01). After treatment with melatonin, there was a statistically significant decrease of the gingival index (15.84 ± 10.3 vs 5.6 ± 5.1) and pocket depth (28.3 ± 19.5 vs 11.9 ± 9.0) (P < 0.001). Also, use of melatonin was associated with a significant reduction of the four biomarkers. Changes of salivary acid phosphatase and osteopontin correlated significantly with changes in the gingival index, whereas changes of alkaline phosphatase and osteopontin correlated significantly with changes in the pocket depth., Conclusions: Treatment with topical melatonin was associated with an improvement in the gingival index and pocket depth, a reduction in salivary concentrations of acid phosphatase, alkaline phosphatase, osteopontin and osteocalcin.

Quercetin and rutin are common flavonoids in fruit and vegetables, and have been reported to affect bone development. However, the effect of flavonoids on osteoblast differentiation remains a matter of controversy. In the present study, mouse bone marrow mesenchymal stem cells (BMMSCs) were isolated and characterized for their use in osteoblast differentiation using two flavonoids, quercetin and rutin. BMMSCs were cultured in various concentrations of quercetin and rutin during the osteoblast differentiation period of 10 days. Both quercetin and rutin were found to up regulate the osteoblast differentiation in dose dependent manner, albeit to lesser extent in case of former than that of latter. Quercetin and rutin also increased alkaline phosphatase activity by about 150 and 240% and demonstrated mineralization up to 110 and 200% respectively as compared to control (which was considered as 100%). Further, both the flavonoids were also found to increase the expression of some of the prominent markers for differentiation of osteoblast like osteopontin, osterix, RunX2, osteoprotegerin and osteocalcin. The current data suggests that certain classes of flavonoids like rutin and quercetin can be used in the cure and management of osteodegenerative disorders due to their osteoblast specific differentiation activities., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Recent studies show that osteopontin (OPN) and its receptor cluster of differentiation 44 (CD44) are two pro-inflammatory cytokines contributing to the development of atherosclerosis. The objective of this study was to explore the inhibitory effect of kaempferol, a naturally occurring flavonoid compound, on atherogenesis and the mechanisms involved. The experiments were performed in aorta and plasma from C57BL/6J control and apolipoprotein E-deficient (ApoE(-/-)) mice treated or not with kaempferol (50 or 100mg/kg, intragastrically) for 4 weeks. Kaempferol treatment decreased atherosclerotic lesion area, improved endothelium-dependent vasorelaxation, and increased the maximal relaxation value concomitantly with decrease in the half-maximum effective concentration, plasma OPN level, aortic OPN expression, and aortic CD44 expression in ApoE(-/-) mice. In addition, treatment with kaempferol also significantly decreased reactive oxygen species production in mice aorta. The present results suggest that kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE(-/-) mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. The spectrum of disease ranges from fatty liver to hepatic inflammation, necrosis, progressive fibrosis and hepatocellular carcinoma. In developed countries, ALD is a major cause of end-stage liver disease that requires transplantation. The most effective therapy for ALD is alcohol abstinence. However, for patients with severe forms of ALD (that is, alcoholic hepatitis) and for those who do not achieve abstinence from alcohol, targeted therapies are urgently needed. The development of new drugs for ALD is hampered by the scarcity of studies and the drawbacks of existing animal models, which do not reflect all the features of the human disease. However, translational research using liver samples from patients with ALD has identified new potential therapeutic targets, such as CXC chemokines, osteopontin and tumor necrosis factor receptor superfamily member 12A. The pathogenetic roles of these targets, however, remain to be confirmed in animal models. This Review summarizes the epidemiology, natural history, risk factors and current knowledge of the pathogenetic mechanisms of ALD. In addition, this article provides a detailed description of the findings of these translational studies and of the animal models used to study ALD.

Objective: Sodium butyrate (butyric acid; BA) is a major metabolic by-product of the anaerobic periodontopathic bacteria present in subgingival plaque. We examined the effects of BA and/or indomethacin on cell proliferation, the expression of cyclooxygenases (COXs), prostaglandin (PG) receptors (EP1-4), extracellular matrix proteins, such as type I collagen and osteopontin, and PGE(2) production, using ROS17/2.8 cells as osteoblasts., Methods: The rat clonal cell line ROS 17/2.8 was cultured with 0, 10(-5), 10(-4), and 10(-3)M BA in the presence or absence of 0.5 μM indomethacin, for up to 7 days. The expression of COX-1, COX-2, EP1, EP2, EP3, EP4, type I collagen, and osteopontin was examined at the mRNA and protein levels using real-time PCR and Western blotting, respectively. The amount of PGE(2) in the culture medium was measured by ELISA., Results: Proliferation of ROS 17/2.8 cells was not affected by the addition of BA. However, PGE(2) production and the expression of COX-1 and COX-2 increased with the addition of BA. In contrast, indomethacin, an inhibitor of COX, blocked the stimulatory effect of BA. Furthermore, EP2 expression increased with BA treatment, whereas EP1 expression was not affected and the expression of EP3 and EP4 was not detected. The addition of BA also increased the expression of type I collagen and osteopontin. Indomethacin blocked about 50% of the stimulatory effect of BA on type I collagen, whereas it did not block the effect on osteopontin., Conclusions: These results suggest that BA induces PGE(2) production by increasing the expression of COX-1 and COX-2 in osteoblasts, and that an autocrine action of the produced PGE(2), via EP1 or BA-induced EP2, is related to an increase in type I collagen expression by BA., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Unlabelled: In this pilot study, we demonstrated that women with osteopontin (OPN) over-expression show less resistance to postmenopausal osteoporosis than women with normal OPN levels. We hypothesized that the levels of plasma OPN could be used as a treatment indicator for intermittent parathyroid hormone (PTH)-treated menopausal osteoporosis. We demonstrated that plasma OPN levels could be used as a biomarker for early treatment response., Introduction: Animal studies indicate that OPN-deficient mice are resistant to ovariectomy induced osteoporosis. Our pilot study also demonstrated women with OPN over expression may show less resistance to postmenopausal osteoporosis. The role of plasma OPN in PTH1-34-treated osteoporosis remains unclear., Methods: From September 2005 to September 2006, 31 menopausal women over 45 years of age with severe osteoporosis were enrolled in our study. Subjects were treated with PTH1-34 subcutaneously at a dose of 20 μg/day. Plasma OPN levels and BMD of the lumbar spine and hip were measured using ELISA and dual-energy X-ray absorptiometry at baseline, 3, 6, and 9 months. Response to the treatment was assessed by the sequential change in bone mineral density and OPN expression using a general linear mixed model., Results: The plasma OPN decreased sequentially and significantly throughout the 9-month treatment course from 20.75 ± 5.36 to 11.2 ± 4.37 ng/ml (p < 0.001). The sequential improvement in the T-score and Z-score was significant in the lumbar spine but not in the hip area. In the lumbar spine, when the plasma OPN decreased by 1 ng/ml the T-score increased by 0.0406 and the Z-score increased by 0.0572 of lumbar spine., Conclusion: OPN levels are related to the anabolic effect of PTH in human postmenopausal osteoporosis. Plasma OPN levels could be used as a biomarker for early treatment response.

Osteopontin (OPN) is a pleiotropic protein implicated in various inflammatory responses including ischemia-reperfusion (I-R) injury. Two distinct forms of the protein have been identified: an extensively studied secreted form (sOPN) and a less-well-known intracellular form (iOPN). Studies have shown that increased OPN expression parallels the time course of macrophage infiltration into injured tissue, a late event in the development of cerebral infarcts. sOPN has been suggested to promote remodeling of the extracellular matrix in the brain; the function of iOPN may be to facilitate certain signal transduction processes. Here, we studied OPN expression in adult male Sprague-Dawley rats subjected to global forebrain I-R injury. We found iOPN in the cytoplasm of both cortices and the hippocampus, but unexpectedly only the right cortex exhibited a marked increase in the iOPN level after 45 min of reperfusion. Acetaminophen, a drug recently shown to decrease apoptotic incidence, caspase-9 activation, and mitochondrial dysfunction during global I-R, significantly inhibited the increase in iOPN protein in the right cortex, suggesting a role for iOPN in the response to I-R injury in the right cortex.

Poor prognosis in ovarian clear cell carcinoma is associated with the expression of a defined set of proteins including osteopontin (OPN) and integrin. Statins, a family of 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, are currently being investigated for the treatment and prevention of cancer. In this study, we investigated the effects of simvastatin on ovarian clear cell carcinoma (OCCC) cells in vitro and in vivo and elucidated the mechanism of drug action. Changes in OPN gene expression were determined by real-time RT-PCR, and an MTT assay was performed to determine effects on cell proliferation. Finally, a xenograft tumor model was constructed to evaluate the effects of simvastatin on cell proliferation and apoptosis in vivo. According to our experimental results, OPN is an important protein in OCCC. Simvastatin inhibited OCCC cell proliferation, and the inhibition rate was approximately 40% to 50% after treatment with 10 µM simvastatin for 48 h. In the xenograft studies, simvastatin treatment resulted in a significant growth inhibition. Furthermore, the mice treated with simvastatin survived significantly longer compared to the control groups. In conclusion, simvastatin has anticancer effects in vitro and in vivo. Further confirmation of the anticancer effects of statins in future studies will increase the scope for OCCC treatment.

Objectives: Tobacco smoking has been suggested to be one of the important risk factors of developing periodontal disease. Although epidemiological studies have shown the detrimental effects of smoking on periodontal disease, the effects of smoke compounds on gingival tissue are not well understood. The aim of this study was to evaluate the effects of nicotine, which is the major component of the thousands of chemicals that constitute cigarette smoke, for cytodifferentiation of murine periodontal ligament (MPDL) cell., Materials and Methods: Expression of nAChR subunits on MPDL cells was examined using RT-PCR. The effects of nicotine on gene expression of extracellular matrices and osteoblastic transcription factors were evaluated by quantitative RT-PCR. Mineralized nodule formation of nicotine-treated MPDL cells was characterized by alizarin red staining., Results: Murine periodontal ligament cells expressed several subunits of nAChR, which have functional calcium signals in response to nicotine. Gene expression of extracellular matrices and osteoblastic transcription factors were reduced in nicotine-treated MPDL cells. In addition, mineralized nodule formation was inhibited in MPDL cells in the presence of nicotine., Conclusion: Our findings indicate that nicotine may negatively regulate the cytodifferentiation and mineralization of MPDL cells., (© 2010 John Wiley & Sons A/S.)

Background: Bone morphogenetic protein (BMP)-7 is a potent bone-inducing factor and was shown to promote periodontal regeneration in vivo and in vitro; however, to our knowledge, the specific effect of BMP-7 on cementoblasts has not been defined. We aimed to investigate the effects of BMP-7 on cementoblasts, which are cells responsible for tooth root-cementum formation. We hypothesized that BMP-7 would regulate mineralized tissue-associated genes in cementoblasts and influence the expression profile of genes associated with cementoblast extracellular matrix (ECM) and cell adhesion molecules (CAMs)., Methods: A murine immortalized cementoblast cell line (OCCM.30) was cultured with and without 50 ng/ml BMP-7. After 72 hours, total RNA was isolated, and mRNA levels for bone/cementum markers, including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor-2 (Runx2), were investigated by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). In vitro mineral nodule formation was assayed on day 8 using von Kossa staining. A pathway-specific gene-expression array was used to determine BMP-7-responsive ECM and CAM genes in cementoblasts., Results: Mineralized tissue markers were strongly regulated by BMP-7, with an almost three-fold increase in BSP and OCN transcripts and significant increases in OPN and Runx2 mRNA expressions. BMP-7 treatment markedly stimulated cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. BMP-7 treatment altered the OCCM.30 expression profile for ECM and CAM functional gene groups. BMP-7 tended to increase the expression of collagens and matrix metalloproteinases (MMPs), mildly decreased tissue inhibitors of MMPs (TIMPs), and had mixed regulatory effects on integrins. Using Q-PCR, selected array results were confirmed, including a significant BMP-7-induced increase in MMP-3 and a decrease in TIMP-2 mRNA expression., Conclusion: These results support the promising applications of BMP-7 in therapies aimed at regenerating periodontal tissues lost as a consequence of disease.

The possibility that dietary vitamin D(3) (VD(3)) exposure inhibits endometrial carcinogenesis in an animal model and modifies the enhanced risk of endometrial carcinoma associated with obesity was investigated. At 4 weeks of age, Pten(+/-) and wild-type mice were each divided into four treatment groups and fed AIN93G control diet, or AIN93G-based diet containing either 25,000 international units of VD(3) per kilogram of diet, 58% fat to induce obesity (high fat), or high fat and 25,000 international units of VD(3) per kilogram of diet. Mice were kept on these diets until they were sacrificed at week 28. Although VD(3) did not affect endometrial cancer risk, it inhibited obesity-induced increase in endometrial lesions. Specifically, high-fat diet increased focal glandular hyperplasia with atypia and malignant lesions from 58% in the control diet-fed Pten(+/-) mice to 78% in obese mice. Dietary VD(3) decreased the incidence of endometrial pathology in obese Pten(+/-) mice to 25% (P < 0.001). VD(3) altered the endometrial expression of 25-hydroxylase, 1α-hydroxylase, and vitamin D receptor in the wild-type and Pten(+/-) mice. Estrogen receptor-α mRNA levels were higher (P < 0.014) and progesterone receptor protein levels in the luminal epithelium were lower (P < 0.04) in the endometrium of control diet-fed Pten(+/-) than wild-type mice, but the expression of these receptors was not affected by the dietary exposures. VD(3) reversed the obesity-induced increase in osteopontin (P < 0.001) and significantly increased E-cadherin expression (P < 0.019) in the endometrium of obese Pten(+/-) mice. Our data confirm the known association between obesity and endometrial cancer risk. Dietary exposure to VD(3) inhibited the carcinogenic effect of obesity on the endometrium. This protective effect was linked to a reduction in the expression of osteopontin and increase in E-cadherin., (©2010 AACR.)

Introduction: Pulp stones are frequently formed as a pathologic calcification product in dental pulp tissues, but the pathogenesis is poorly understood. We previously found that osteopontin (OPN) was produced by dental pulp cells, and its expression was associated with formation of the pulp stone matrix. It was reported that amorphous calcification appeared in the dental pulp of diabetic patients. The aim of this study was to determine the relationship between OPN expression and pathologic calcification in rat diabetic pulp., Methods: The effect of glucose on OPN production and alkaline phosphatase activity in cultured rat dental pulp cells (RPC-C2A) was investigated, and then dental pulp calcification and OPN expression in diabetic rats were determined and compared with those in healthy rats by histologic and immunohistochemical analyses., Results: In RPC-C2A cells, biochemical analysis showed that a high concentration of glucose (50 mmol/L) increased OPN protein production and alkaline phosphatase activity 1.3-fold and 1.5-fold, respectively. Histologic observations showed more calcified particles in dental pulp tissues in diabetic than in nondiabetic rats. Moreover, a thickened layer of predentin was formed in the radicular pulp of diabetic rats. OPN was more strongly stained around the calcified particles and in the odontoblast zone under the thickened predentin in diabetic rats., Conclusions: OPN might be a key molecule involved in the increase of pathologic pulp calcifications, which are frequently observed in diabetic patients., (Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background and Objective: Malassez's epithelial rest (MER) cells are involved in the maintenance and homeostasis of the periodontal ligament (PDL). The purpose of this study was to determine the effects of epidermal growth factor (EGF) and/or nerve growth factor (NGF) in vitro on these functions of MER cells., Material and Methods: MER cells from porcine PDL were incubated for 3 or 9 h after the addition of EGF and/or NGF to final concentrations of 10 ng/mL. Cells cultured without those growth factors were used as controls. The expression of mRNA for osteopontin, bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) was analyzed using quantitative RT-PCR., Results: There was a decrease in the expression of osteopontin mRNA by MER cells treated for 9 h with NGF and the level of mRNA expressed was lower than that of the control and EGF-treated groups. The expression of BMP-2 mRNA by MER cells treated with NGF for 9 h also decreased, and was lower than that of the control and EGF-treated groups. The expression of VEGF mRNA by MER cells treated with EGF for 3 or 9 h was higher than in the control and NGF-treated groups. The expression of VEGF mRNA was lower in MER cells treated with NGF for 3 and 9 h than in the control and EGF-treated groups, and decreased from 3 to 9 h of treatment. EGF stimulated MER cells to secrete VEGF, which suggests that EGF plays an important role in maintaining the homeostasis of the PDL. NGF acts on MER cells to inhibit calcification in the PDL. Furthermore, in the EGF+NGF-treated MER cells, expression of mRNA for BMP-2 and VEGF was similar to that of the NGF-treated group, but cell proliferation and expression of osteopontin mRNA were similar to that of the EGF-treated group., Conclusion: EGF and NGF play important roles in maintaining the PDL.

Background and Objective: Our previous study found that thrombin induced osteoprotegerin synthesis in human periodontal ligament cells. As elevated levels of osteoprotegerin can exert biological effects on various cell types, in the present study we investigated the effect of osteoprotegerin on the expression of osteopontin in human periodontal ligament cells., Material and Methods: Cultured human periodontal ligament cells were treated with recombinant human osteoprotegerin (0-100 ng/mL) for 24-48 h. The expression of osteopontin mRNA and protein was analyzed using reverse transcription-polymerase chain reaction and western blot analyses, respectively. Phosphoinositol 3-kinase inhibitor, Akt inhibitor, heparinase, neutralizing antibody against receptor activator of nuclear factor-kappaB ligand (RANKL) and syndecan-1, and small interfering RNA against syndecan-1, were used to determine the mechanism involved., Results: Osteoprotegerin up-regulated the mRNA and protein expression of osteopontin in human periodontal ligament cells in a dose-dependent manner. Addition of neutralizing antibody against RANKL attenuated the inductive effect of osteoprotegerin on osteopontin expression. In addition, the inductive effect of osteoprotegerin was abolished by phosphoinositol 3-kinase and Akt inhibitors, as well as by syndecan-1 antibody or syndecan-1 small interfering RNA. None of the inhibitors had any effect on the background level of osteopontin expression., Conclusion: An increased level of osteoprotegerin can generate signals via a RANKL/syndecan-1/phosphoinositol 3-kinase/Akt pathway. The results also suggest that osteopontin is one of the downstream targets of the pathway mediated by osteoprotegerin in human periodontal ligament cells. Thus, in addition to counteracting RANKL in the RANKL-osteoprotegerin system, osteoprotegerin may play a role in periodontal tissue remodeling through modulation of the extracellular matrix.

Background: This study examined the effects of nicotine on osteoblastic differentiation and the osteoclastogenesis regulatory molecules receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). In addition, we investigated the mechanism by which nicotine induced antioxidant defense enzyme expression as a protective response., Methods: The expression of osteoblast markers, RANKL, OPG, and antioxidant defense enzymes were examined in nicotine-treated human periodontal ligament (PDL) cells by reverse transcription-polymerase chain reaction and Western blotting., Results: Nicotine treatment concomitantly downregulated the expression of OPG and osteoblastic differentiation markers, such as alkaline phosphatase, osteocalcin, and osteopontin, and upregulated the expression of RANKL. Nicotine induced the synthesis of the transcription factor NF-E2-related factor-2 (Nrf2) as well as a number of cellular antioxidants and phase II enzymes, such as heme oxygenase-1. Pretreatment with antioxidants inhibited the upregulation of RANKL, the downregulation of OPG expression, and cytotoxicity by nicotine in PDL cells., Conclusions: Nicotine upregulated RANKL and antioxidant defense enzymes. These data suggest that Nrf2-mediated induction of cellular antioxidants and phase II enzymes could contribute to the cellular defense against nicotine-induced cytotoxicity and osteoclastic differentiation in PDL cells.

Objective: The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation., Methods: Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR)., Results: We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation., Conclusions: The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.

Avian species have the possible risk of embryonic exposure to persistent, lipophilic environmental contaminants, such as dichlorodiphenyltrichloroethane (DDT), by transfer of chemicals accumulated in mother birds to eggs. To model developmental and reproductive disorders of wild birds living in contaminated areas, we exposed Japanese quails in ovo to o,p'-DDT prior to incubation. A positive estrogenic substance diethylstilbestrol (DES; 1 and 10 ng/g of egg) and o,p'-DDT (1-100 microg/g of egg) were injected into the yolk before incubation. Treatment with o,p'-DDT (10 or 100 microg/g) but not with DES significantly reduced the hatchability of eggs. After sexual maturation, o,p'-DDT affected eggshell formation in female quails but had little influence on laying; high doses of o,p'-DDT significantly reduced eggshell strength, shell weight, and shell thickness, and several females treated with 100 microg o,p'-DDT/g laid eggs lacking shells. Diethylstilbestrol decreased egg production itself but had little effect on the eggshell. Both o,p'-DDT and DES caused dose-dependent shortening of the left oviduct and abnormal development of the right oviduct in females, while testis asymmetry was observed in males treated with a high dose of DES. In the uterus of the oviduct, the mRNAs for calcium-regulating factors osteopontin and calbindin D28K were reduced by both treatments, particularly that with o,p'-DDT. The results indicated that transovarian exposure to o,p'-DDT could bring about population declines in avian species through loss of fecundity caused by depression of hatchability and dysfunction of the reproductive tract.

Purpose: The use of nanotechnology to enhance endosseous implant surfaces may improve the clinical control of interfacial osteoblast biology. This study investigated the influence of a nanostructure-coated implant surface on osteoblast differentiation and its effects on bone-to-implant contact (BIC) and removal torque values., Materials and Methods: Titanium disks were machined (M) or machined and subsequently treated by acid etching (Ac) or by dipping in an aluminum oxide solution (Al2O3). Surfaces were characterized by scanning electron microscopy, atomic force microscopy, and x-ray microanalysis. For the in vitro experiment, rat mesenchymal stem cells (rMSCs) were grown in osteogenic supplements on the disk surfaces for 3 days. Real-time polymerase chain reaction (PCR) was used to measure mRNA levels of several gene products (bone sialoprotein, osteocalcin, osteopontin, and RUNX-2). For the in vivo experiment, titanium implants were placed in rat tibiae and harvested after 3 to 21 days for measurement of bone-specific mRNA levels by real-time PCR. Removal torque and BIC were measured 3 to 56 days after placement., Results: Average height deviation (Sa, in nm) values for M, Ac, and Al2O3 implants were 86.5, 388.4, and 61.2, respectively. Nanostructured Al2O3 topographic features applied to machined implants promoted MSC commitment to the osteoblast phenotype. Greater bone-specific gene expression was observed in tissues adjacent to Al2O3 implants, and associated increases in BIC and torque removal were noted., Conclusion: Nanostructured alumina may directly influence cell behavior to enhance osseointegration.

Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in cardiovascular diseases. Isorhynchophylline, an alkaloid from a traditional Chinese medicine Gambirplant, has been used to treat cardiovascular diseases. The aim of this study was to investigate the effects of isorhynchophylline on angiotensin II (Ang II)-induced proliferation of rat VSMCs. VSMCs were isolated from rat artery and cultured for 14 days before experimentation. The effect of isorhynchophylline on Ang II-induced proliferation was evaluated by cell number, MTT assay and flow cytometry, and nitric oxide (NO) content and activity of NO synthase (NOS) were measured. The expression of proto-oncogene c-fos, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) mRNAs was measured by real-time RT-PCR. VSMC cultures were verified by morphology and immunostaining with alpha-smooth muscle actin. Isorhynchophylline (0.1-10.0 microM) was not toxic to VSMCs, but markedly decreased Ang II (1.0 microM)-enhanced cell number and MTT intensity, and blocked cell transition from G(0)/G(1) to S phase. Furthermore, isorhynchophylline increased the NO content and NOS activity, and suppressed Ang II-induced over-expression of c-fos, OPN and PCNA. Thus, isorhynchophylline was effective against Ang-II induced cell proliferation, an effect that appears to be due, at least in part, to increased NO production, regulation of the cell cycle, and depressed expression of c-fos, OPN and PCNA related to VMSC proliferation.

Objective: The purpose of this study was to determine the effects of enamel matrix derivative on mRNA expression of markers related to periodontal healing., Study Design: Murine osteoprogenitor cells (MC3T3-E1) were grown for 12 and 16 days in mineralization media and stimulated with 100 microg/mL Emdogain (EMD). Cell cultures treated with 2% and 10% fetal calf serum (FCS) served as control. The mRNA expression of bone sialoprotein (BSP), osteopontin (OPN), and runt-related protein 2 (Runx2) was analyzed by real-time polymerase chain reaction. One-way analysis of variance was used for statistical analysis., Results: Stimulation with EMD significantly (P < .01) enhanced mRNA expression of BSP up to 13.9-fold and of OPN up to 3.2-fold at day 16 compared with the 2% FCS control. The expression of mRNA for transcription factor Runx2 was not significantly changed., Conclusion: The beneficial effects seen in periodontal regeneration after treatment with EMD may be related to an increase of the mineralization markers BSP and OPN at mRNA level.