Bone Sialoprotein Gene Expression Research Articles

The potential use of ascorbic acid to recover the cellular senescence of lipopolysaccharide-induced human apical papilla cells: an in vitro study.

To examine the effect of lipopolysaccharide (LPS) on cellular senescence induction of human apical papilla cells (hAPCs) and evaluate the potential use of 50 μg/ml ascorbic acid to recover cellular senescence and regenerative functions. hAPCs were treated with LPS at 1 and 10 μg/ml either with or without 50 μg/ml ascorbic acid for 48 h. The cellular senescence biomarkers were analyzed by senescence-associated β-galactosidase (SA-β-gal) staining and senescence-related gene expression, p16 and p21. Cell migration, at 12 h and 24 h, was evaluated using a scratch wound assay. Mineralization potential was assessed at 21 days using Alizarin red S staining and dentine sialophosphoprotein (DSPP) and bone sialoprotein (BSP) gene expression. 1 μg/ml and 10 μg/ml LPS stimulation for 48 h induced cellular senescence, as shown by remarkable SA-β-gal staining and p16 and p21 gene expression. The percentage of wound closure and mineralized formation was reduced. The co-incubation with ascorbic acid significantly down-regulated the level of SA-β-gal staining. The reduction of senescence-associated gene expressions was observed. Ascorbic acid improved cell migration, mineralized nodule formation, and the expression of DSPP and BSP genes in LPS-treated hAPCs. LPS significantly promoted cellular senescence on hAPCs and diminished the cell function capacity. Co-presence of ascorbic acid could impede cellular senescence and possibly improve the regenerative capacity of LPS-induced senescent hAPCs in vitro. The data support the in vitro potential benefit of ascorbic acid on cellular senescence recovery of apical papilla cells.

Sema4D is diminished in leukocyte platelet-rich fibrin and impairs pre-osteoblastic MC3T3-E1 cells' functionality.

ObjectiveSemaphorin 4D (Sema4D) is a coupling factor expressed on osteoclasts that may hinder osteoblast differentiation. Since the leukocyte platelet-rich fibrin (L-PRF) membrane promotes growth factor concentration, this study aims to quantify the amount of Sema4D in L-PRF membranes, and analyze the impact of Sema4D on osteoblast cell function in vitro. DesignEnzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of Sema4D in both L-PRF and whole blood (serum). To analyze the impairment of Sema4D on osteoblasts, MC3T3-E1 cells were induced to osteogenic differentiation and exposed to Sema4D ranging from 10 to 500 ng/ml concentrations. The following parameters were assayed: 1) cell viability by MTT assay after 24, 48, and 72 h; 2) matrix mineralization by Alizarin Red staining after 14 days, 3) Runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), osteonectin (ONC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) gene expression by qPCR. For all data, the significance level was set at 5%. ResultsThe amount of Sema4D in the whole blood (serum) was higher than in L-PRF. Osteoblasts exposed to Sema4D at all tested concentrations exhibited a decrease in matrix mineralization formation as well in RUNX-2, OCN, ONC, BSP, and ALP gene expression (p < 0.05). ConclusionThe presence of Sema4D, a molecule known for suppressing osteoblast activity, diminishes within L-PRF, enhancing its ability to facilitate bone regeneration.

Effects of phase-transited lysozyme on adhesion, migration and odontogenic differentiation of human dental pulp cells: An in vitro study.

To explore the effects of phase-transited lysozyme (PTL) coated dentine slices on cell adhesion, migration and odontogenic differentiation of human dental pulp cells (HDPCs). Cell growth and cell cycle analysis were conducted to verify the biocompatibility of PTL for HDPCs. Cell adhesion, cell morphology and proliferation were explored by DiI staining, Scanning electron microscopy and MTT assay. Cell migration was investigated by Transwell assay. The effects of PTL on odontogenesis and mineralization of HDPCs were assessed by real-time quantitative polymerase chain reaction and Western blot. The mineralization of HDPCs was evaluated by Alizarin red staining. HDPCs were isolated from extracted third molars. The level of statistically significant difference was accepted at p < .05. PTL showed no negative effect on cell cycle of HDPCs and compared with the blank group, HDPCs labelled with DiI staining showed significantly more adhered cells at 48 h (p < .05), extending cell processes and more finger-like or reticular pseudopodia on PTL-coated dentine slices. The results of MTT and Transwell assay showed that PTL promoted the proliferation (p < .05) and migration (p < .01) of HDPCs, respectively. Compared with the blank group, the gene expression of dentine sialophosphoprotein (DSPP), osteopontin and bone sialoprotein in HDPCs cultured on PTL was significantly upregulated on day 3 and 7 (p < .05), while the protein expression of DSPP showed no significant change on both day 7 and day 14. Alizarin red staining showed that PTL promoted more mineralization nodules formation of HDPCs (p < .05). PTL promoted the adhesion, proliferation and migration of HDPCs on dentine slices, and positively affected odontogenic differentiation and mineralization of HDPCs.

Effect of Er: YAG Laser Irradiation on Bone Metabolism-Related Factors Using Cultured Human Osteoblasts.

Introduction: A variety of laser treatments have been applied in numerous medical fields. In dentistry, laser treatments are used for caries, root canals, and periodontal disease, as well as surgical resection. Numerous reports have recently been published on the use of lasers for bone regeneration. If laser irradiation is found to promote the activation of bone metabolism, it might also be effective for periodontal treatment, peri-implantitis, and bone regeneration. Therefore, the present in vitro study aimed to elucidate the mechanisms underlying the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on the bone using osteoblast-like cells. Methods: Osteoblast-like Saos 2 cells (5.0×104 cells) were seeded in 24-well plates. 24 hours after being seeded, the cells were subjected to 0.3 W, 0.6 W, and 2.0 W Er: YAG laser irradiation and then allowed to recover for 48 hours. The expression levels of bone metabolism-related factors alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteoprotegerin (OPG) were then evaluated using reverse transcription-quantitative polymerase chain reaction and western blot analyses. Results: Saos 2 cells subjected to Er: YAG laser irradiation at 0.3 W, 0.6 W, and 2.0 W showed normal growth. When the Er: YAG laser irradiation and control groups were compared after 48 hours, increases were observed in ALP, BSP, and OPG gene and protein expression in the 2.0 W group. Similar results were obtained in the western blot analysis. Conclusion: These findings suggest that the Er: YAG laser irradiation of osteoblast-like cells is effective for activating bone metabolism factors.

Human Non-Hypertrophic Nonunion Tissue Contains Osteoblast Lineage Cells and E-BMP-2 Activates Osteogenic and Chondrogenic Differentiation.

In this study, we examined the proliferation capability and osteogenic and chondrogenic differentiation potential of non-hypertrophic nonunion cells (NHNCs), and the effect of Escherichia coli-derived BMP-2 (E-BMP-2) on them. We enrolled five patients with non-hypertrophic nonunion. NHNCs isolated from nonunion tissue sampled during surgery were cultured, passaged, counted every 14 days, and analyzed. NHNCs were homogenous fibroblastic adherent cells and long-lived through at least 10 passages, with a slight decline. The cells were consistently positive for mesenchymal stem cell-related markers CD73 and CD105, and negative for the hematopoietic markers CD14 and CD45. NHNCs could differentiate into osteoblast lineage cells; however, they did not have strong calcification or sufficient chondrogenic differentiation capability. E-BMP-2 did not affect the proliferative capability of the cells but improved their osteogenic differentiation capability by increasing alkaline phosphatase activity and upregulating the gene expression of osterix, bone sialoprotein, and osteocalcin. E-BMP-2 enhanced their chondrogenic differentiation capability by upregulating the gene expression of aggrecan and collagen type II. We showed, for the first time, that NHNCs have the capacity to differentiate into osteoblast-lineage cells, although the chondrogenic differentiation potential was poor. Local application of E-BMP-2 with preservation of nonunion tissue is a potential treatment option for non-hypertrophic nonunion.

Adoption of Biomedical Ceramic iRoot BP in the Treatment of Localized Pulpitis in Children

To explore the clinical efficacy of biomedical ceramic iRoot BP in the treatment of localized acute pulpitis in children, and the effect of iRoot BP on proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs), 72 localized acute pulpitis children admitted to our hospital from September 2018 to September 2019 were selected and divided into group A (treated with MTA pulp capping material) and group B (treated with iRoot BP material), and the clinical effect, pain degree, and adverse reactions (ADR) rate were compared. The effects of iRoot BP on hDPSCs proliferation and osteogenic differentiation were analyzed; the proliferative activity of cells in iRoot BP group, MTA group, and control group (C group) were measured by cholecystokinin-8 (CCK-8) assay, the ability of cell mineralized nodular formation was observed via alizarin red staining; and quantitative reverse transcription PCR (qRT-PCR) andWestern blot were adopted to determine the expression of osteogenic related genes of hDPSCs and key proteins of mitogen-activated protein kinase (MAPK) signaling pathway. After 1 week of treatment, the clinical efficacy of group B was more favorable in contrast with group A (P &lt; 0.05); the pain of children in group B was notably better in contrast with group A, and incidence of ADR was notably lower in contrast with group A (P &lt; 0.05). 5.0 mg/mL, 10.0 mg/mL, and 30 mg/mL iRoot BP or MTA could improve cell proliferation activity (P &lt; 0.01); the effect of iRoot BP on proliferation of hDPSCs was greater in contrast with MTA (P &lt; 0.05); and the integral optical density (IOD) value of iRoot BP group was notably higher in contrast with MTA group (P &lt; 0.01). The mRNA expression levels of collagen-I (COL-I), bone sialoprotein (BSP), and osteocalcin (OC) in MTA group and iRoot BP group were notably higher in contrast with C group (P &lt; 0.01); the COL-I mRNA expression level of iRoot BP group was notably higher in contrast with MTA group (P &lt; 0.01); the mRNA expression level of BSP in MTA group was notably higher in contrast with iRoot BP group (P &lt; 0.01); the relative protein expression levels of phosphorylated ERK (p-ERK) and phospho-Jun N-terminal kinase (p-JNK) in MTA group and iRoot BP group were notably higher in contrast with C group (P &lt; 0.01); and the relative expression level of p-ERK protein in iRoot BP group was higher in contrast with MTA group (P &lt; 0.05). These results indicated that the clinical efficacy of biomedical ceramic iRoot BP was better than MTA in the preservation of live pulpitis in children, and the patients treated with iRoot BP had better pain recovery effect and lower risk of ADR. The effect of iRoot BP on the proliferation and mineralization of hDPSCs was better than that of MTA, and it may promote the osteogenic differentiation of hDPSCs by activating MAPK signaling pathway and regulating gene expression of COL-I, BSP, and OC.

Interactions between cadmium and zinc on gene expression pattern of differentiation markers in MC3T3-E1 cell line

We evaluated, in vitro, the interactions between cadmium (Cd) and zinc (Zn) during the proliferation and differentiation process using bone MC3T3-E1 cell line. Cells were treated with CdCl2 and/or ZnCl2 for 24 and 48 h and 5 µM CdCl2 was found as low cytotoxic dose and 25 µM ZnCl2 as the best Zn treatment for cell proliferation. Gene expression of some bone markers (Runx2, collagen α1 (Colα1), osteocalcin (Oc), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) was studied at 24, 48 and 72 h. Treatment by CdCl2 depressed Runx2, Colα1, and BSP mRNA levels after 24 h. Oc and ALP gene expression was found to be decreased after 72 h. CdCl2 -exposure decreased ALP activity and Ca deposit in matrix. In concomitant treatment by CdCl2 and ZnCl2, gene expression of osteoblastic markers was found to be up-regulated (p < 0, 05) compared to CdCl2 treated cells, ALP staining and mineralization were increased. Our results show that Zn could prevent Cd-induced toxicity on MC3T3-E1 cells, probably through the restoration of Runx2, col α1, BSP, ALP and Oc and gene expression inhibited by Cd.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

ObjectiveTo investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro.Materials and MethodsRecombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis.ResultsThe prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis.ConclusionsCo-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Effects of lipopolysaccharide on proliferation, migration and osteogenic differentiation of apical papilla cells from early and late stage of root development.

The aim of this study was to investigate the effects of lipopolysaccharide on cell proliferation, migration and osteogenic differentiation of apical papilla cells from early and late stage of root development. After challenging with various lipopolysaccharide concentrations to apical papilla cells from both stages of root development for 168h, cell proliferation and migration were investigated. Osteogenic differentiation was examined by Alizarin red staining, and gene expressions of bone/cementum or dentin-related genes were examined by polymerase chain reaction. Lipopolysaccharide did not affect cell proliferation and migration in both groups. Lipopolysaccharide at 1 and 5µgmL-1 increased Alizarin red staining in apical papilla cells from early-stage but not the late-stage cells. Bone sialoprotein (bone/cementum marker) gene expression increased in both early and late stage of root development at 5µgmL-1 . These results might explain bone/cementum generation in regenerative endodontic procedures.

Size-Optimized Microspace Culture Facilitates Differentiation of Mouse Induced Pluripotent Stem Cells into Osteoid-Rich Bone Constructs.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 μm (Elp400), 500/700 μm (Elp500), and 900/700 μm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS).Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively.ResultsFAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces.Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Biogenic silica nanostructures derived from Sorghum bicolor induced osteogenic differentiation through BSP, BMP-2 and BMP-4 gene expression

Development of scaffolds from naturally available biomaterials for bone tissue engineering is an interesting research area. Among the different scaffolds established for this purpose, biogenic silica nanostructure (BSN) serves as a promising naturally available inorganic and biocompatible material. A few studies have demonstrated the intrinsic biological activity of synthetic silica nanoparticles. The virtually infinite promising applications of these structures rely on their erratic physicochemical properties. We have derived BSNs from Sorghum bicolor seed head using a progressive approach. The intrinsic biological activities were analyzed using human mesenchymal stem cells (hMSCs) as an in vitro model with MTT assay and acridine orange/ethidium bromide staining. We also studied the role of BSNs in the osteogenic differentiation of hMSCs using alkaline phosphatase staining, alizarin red staining, and gene expression analysis. BSNs increased the formation of calcium nodules and stimulated alkaline phosphatase (ALP) activity. Significant changes and/or upregulation in the expression of osteogenic prominent markers such as ALP, bone morphogenetic protein-2 (BMP-2), BMP-4, bone sialoprotein (BSP), collagen-1 (Col-1), and Runt-related transcription factor 2 (RUNX2) genes were observed. Taken together, these results suggest that BSNs exhibited biocompatibility and induced osteogenic differentiation of hMSCs, indicative of their potential applications for bone tissue engineering.

Induction of Osteogenic Differentiation of Mesenchymal Stem Cells by Bioceramic Root Repair Material.

This study aimed to evaluate the osteogenic activity of Endosequence Root Repair Material (ERRM) putty using rat mesenchymal stem cells (MSCs). The extract of set ERRM and ProRoot-mineral trioxide aggregate (MTA) (control) was cocultured with rat MSCs and incubated for one, three, and seven days. The cell viability and proliferation were assessed. A quantitative real-time polymerase chain reaction for bone morphogenetic protein-2 (BMP-2), alkaline phosphatase, bone sialoprotein, and osteocalcin gene expression was performed. Both materials enhanced cell viability and proliferation, which increased over time. On day seven, the cells treated with either material exhibited significantly greater cell viability compared with control untreated cells. MSCs treated with either material showed deeper alkaline phosphatase staining after three days compared to control untreated cells. Treated MSCs also exhibited upregulation of the gene expression of bone morphogenetic protein-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. Both ERRM and ProRoot-MTA enhance the osteogenic differentiation of MSCs.

Inhibiting effect of microRNA-187-3p on osteogenic differentiation of osteoblast precursor cells by suppressing cannabinoid receptor type 2

Induction of osteoblast differentiation is an effective approach in promoting osteoblastogenesis and bone formation. MicroRNA (miR) is a kind of small regulatory RNA molecules that control both physiological and pathological processes. The purpose of this research was to explore the function of miR-187-3p in proliferation and osteogenic differentiation in human osteoblastic precursor cells (hFOB 1.19). Our results showed a significant promotion of cell proliferation by miR-187-3p in hFOB 1.19 cell accompanied by increased proliferating cell nuclear antigen (PCNA) and Ki67 expression, whereas miR-187-3p knockdown led to an inhibition of cell proliferation. Moreover, our data revealed that miR-187-3p was decreased in hFOB 1.19 cells undergoing osteoblastic differentiation. Silencing of miR-187-3p dramatically accelerated hFOB 1.19 osteoblastic differentiation, as evidenced by the increase of alkaline phosphatase (ALP) activity and calcium deposition, as well as elevated osteopontin (OPN), collagen type I alpha 1 (COL1A1), and bone sialoprotein (BSP) gene expression, whereas overexpression of miR-187-3p suppressed osteoblastic differentiation. Furthermore, we demonstrated that miR-187-3p could inhibit cannabinoid receptor type 2 (CNR2) expression by targeting its 3’ untranslated region (UTR). Upregulation of CNR2 inversed the inhibiting influence of miR-187-3p on hFOB 1.19 osteogenic differentiation. Collectively, our results showed a pivotal role of miR-187-3p/CNR2 axis in osteoblastic differentiation, indicating that miR-187-3p may serve as a promising target in the therapy of osteoporosis.

Activation of local bone RAS by maternal excessive glucocorticoid participated in the fetal programing of adult osteopenia induced by prenatal caffeine exposure

This study was aimed to investigate whether and how prenatal caffeine exposure (PCE) could induce osteopenia in the adult offspring. Pregnant rats were treated with prenatal caffeine 12 mg/100 g body weight per day from pregnant day 9 to 20, while rat bone marrow mesenchymal stem cells (BMSCs) were treated with exogenous corticosterone during osteogenic induction. Shorter femur and primary ossification center was observed in the PCE offspring, as well as less bone trabecular and poor biomechanical intensity. Local gene expression of glucocorticoid receptor (GR) and angiotensin converting enzyme (ACE), as well as angiotensin 2 content, was found to be stimulated, while the expression of bone gamma-carboxyglutamate protein (BGLAP), alkaline phosphatase (ALP) and bone sialoprotein (BSP) was found to be suppressed, with hypomethylation of ACE promoter. Corticosterone (1250 nM) suppressed osteogenic differentiation of BMSCs and gene expression of BGLAP, ALP and BSP, which was attenuated by enalapril, while it stimulated ACE mRNA expression and induced hypomethylation of ACE promoter, which was attenuated by mifepristone. It indicated that PCE caused bone growth retardation and adult osteopenia in offspring, which might be triggered by the activation of local RAS induced by excessive maternal glucocorticoid, while the hypomethylation of ACE gene might be the key point of the sustained activation of the local RAS.

Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts.

Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

Effect of bone morphogenetic protein 9 on osteoblast differentiation of cells grown on titanium with nanotopography.

Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants.

Physical properties and biological effects of mineral trioxide aggregate mixed with methylcellulose and calcium chloride.

Objectives:Methylcellulose (MC) is a chemical compound derived from cellulose. MTA mixed with MC reduces setting time and increases plasticity. This study assessed the influence of MC as an anti-washout ingredient and CaCl2 as a setting time accelerator on the physical and biological properties of MTA.Material and Methods:Test materials were divided into 3 groups; Group 1(control): distilled water; Group 2: 1% MC/CaCl2; Group 3: 2% MC/CaCl2. Compressive strength, pH, flowability and cell viability were tested. The gene expression of bone sialoprotein (BSP) was detected by RT-PCR and real­ time PCR. The expression of alkaline phosphatase (ALP) and mineralization behavior were evaluated using an ALP staining and an alizarin red staining.Results:Compressive strength, pH, and cell viability of MTA mixed with MC/CaCl2 were not significantly different compared to the control group. The flowability of MTA with MC/CaCI2 has decreased significantly when compared to the control (p<.05). The mRNA level of BSP has increased significantly in MTA with MC/CaCl2 compared to the control (p<.05). This study revealed higher expression of ALP and mineralization in cells exposed to MTA mixed with water and MTA mixed with MC/CaCl2 compared to the control (p<.05).Conclusions:MC decreased the flowability of MTA and did not interrupt the physical and biological effect of MTA. It suggests that these cements may be useful as a root-end filling material.

Effects of Wnt3a on osteogenic differentiation of dental pulp stem cells

Objective: To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells (DPSC). Methods: DPSCs were subjected to different concentrations of Wnt3a (0, 5, 20, 50 and 100 μg/L) and at seven days after culture the alkaline phosphatase (ALP) activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type Ⅰ (COL-Ⅰ), Runt-related transcription factor-2 (RUNX2) was examined by quantitative real-time PCR (qPCR). Results: After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity (concentration 0: 1.076±0.203, 5 μg/L: 0.828±0.118, 20 μg/L: 0.505±0.044, 50 μg/L: 0.499±0.038, 100 μg/L: 0.483±0.060). The expression of OCN in 5 μg/L Wnt3a group (0.092±0.005) was lower than that in culture medium (0.858±0.190)(P<0.05). Alizarin red staining showed that 5 μg/L Wnt3a had no mineralization induction effect on DPSC. Conclusions: Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.

Biological and Mechanical Effects of Micro-Nanostructured Titanium Surface on an Osteoblastic Cell Line In vitro and Osteointegration In vivo.

Hybrid micro-nanostructure implant surface was produced on titanium (Ti) surface by acid etching and anodic oxidation to improve the biological and mechanical properties. The biological properties of the micro-nanostructure were investigated by simulated body fluid (SBF) soaking test and MC3T3-E1 cell co-culture experiment. The cell proliferation, spreading, and bone sialoprotein (BSP) gene expression were examined by MTT, SEM, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the mechanical properties were evaluated by instrumented nanoindentation test and friction-wear test. Furthermore, the effect of the micro-nanostructure surface on implant osteointegration was examined by in vivo experiment. The results showed that the formation of bone-like apatite was accelerated on the micro-nanostructured Ti surface after immersion in simulated body fluid, and the proliferation, spreading, and BSP gene expression of the MC3T3-E1 cells were also upregulated on the modified surface. The micro-nanostructured Ti surface displayed decreased friction coefficient, stiffness value, and Young's modulus which were much closer to those of the cortical bone, compared to the polished Ti surface. This suggested much better mechanical match to the surrounding bone tissue of the micro-nanostructured Ti surface. Furthermore, the in vivo animal experiment showed that after implantation in the rat femora, the micro-nanostructure surface displayed higher bonding strength between bone tissues and implant; hematoxylin and eosin (H&E) staining suggested that much compact osteoid tissue was observed at the interface of Micro-nano-Ti-bone than polished Ti-bone interface after implantation. Based on these results mentioned above, it was concluded that the improved biological and mechanical properties of the micro-nanostructure endowed Ti surface with good biocompatibility and better osteointegration, implying the enlarged application of the micro-nanostructure surface Ti implants in future.