ALPase Activity Research Articles

Fabrication of Solvent-Free PCL/β-TCP Composite Fiber for 3D Printing: Physiochemical and Biological Investigation

Manufacturing three-dimensional (3D) objects with polymers/bioceramic composite materials has been investigated in recent years. In this study, we manufactured and evaluated solvent-free polycaprolactone (PCL) and beta-tricalcium phosphate (β-TCP) composite fiber as a scaffold material for 3D printing. To investigate the optimal ratio of feedstock material for 3D printing, the physical and biological characteristics of four different ratios of β-TCP compounds mixed with PCL were investigated. PCL/β-TCP ratios of 0 wt.%, 10 wt.%, 20 wt.%, and 30 wt.% were fabricated, with PCL melted at 65 °C and blended with β-TCP with no solvent added during the fabrication process. Electron microscopy revealed an even distribution of β-TCP in the PCL fibers, while Fourier transform infrared spectroscopy demonstrated that the biomaterial compounds remained intact after the heating and manufacturing process. In addition, adding 20% β-TCP into the PCL/β-TCP mixture significantly increased hardness and Young's Modulus by 10% and 26.5%, respectively, suggesting that PCL-20 has better resistance to deformation under load. Cell viability, alkaline phosphatase (ALPase) activity, osteogenic gene expression, and mineralization were also observed to increase according to the amount of β-TCP added. Cell viability and ALPase activity were 20% higher with PCL-30, while upregulation for osteoblast-related gene expression was better with PCL-20. In conclusion, PCL-20 and PCL-30 fibers fabricated without solvent exhibited excellent mechanical properties, high biocompatibility, and high osteogenic ability, making them promising materials for 3D printing customized bone scaffolds promptly, sustainably, and cost-effectively.

Photobiomodulation therapy at red and near-infrared wavelengths for osteogenic differentiation in the scaffold-free microtissues.

One of the novel strategies for bone tissue regeneration is photobiomodulation (PBM) which depends on the red and near-infrared light absorption by mitochondria and may trigger bone tissue regeneration via the production of intracellular ROS and ATP, NO release, etc. It is also important to identify the changes in those signal molecule levels in an in vivo mimicking platform such as 3-Dimensional (3D) Scaffold Free Microtissues (SFMs) that may serve more natural osteogenic differentiation responses to PBM. Herein, we aimed to increase the osteogenic differentiation capability of the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVECs) on 3D SFMs by triple light treatment at 655 and 808-nm of wavelengths with the energy densities of 1, 3, and 5J/cm2. We performed the analysis of cell viability, diameter measurements of SFMs, intracellular ROS production, NO release, ATP activity, temperature measurements, DNA content, ALPase activity, calcium content, and relative gene expressions of ALP, Collagen, and Osteopontin by qRT-PCR. It was found that both wavelengths were effective in terms of the viability of SFMs. 1 and 5J/cm2 energy densities of both wavelengths increased the SFM diameter with significant changes in intracellular ROS, ATP, and NO levels compared to the control group. We concluded that PBM therapy was successful to induce osteogenesis. 1J/cm2 at 655nm of wavelength and 5J/cm2 at 808nm of wavelength were the most effective energy densities for osteogenic differentiation on SFMs with triple light treatment.

Lipase‐a single‐nucleotide polymorphism rs143793106 is associated with increased risk of aggressive periodontitis by negative influence on the cytodifferentiation of human periodontal ligament cells

Aggressive periodontitis (AgP) is characterized by general health and rapid destruction of periodontal tissue. The familial aggregation of this disease highlights the involvement of genetic factors in its pathogeny. We conducted a genome-wide association study (GWAS) to identify AgP-related genes in a Japanese population, and the lipid metabolism-related gene, lipase-a, lysosomal acid type (LIPA), was suggested as an AgP candidate gene. However, there is no report about the expression and function(s) of LIPA in periodontal tissue. Hence, we studied the involvement of how LIPA and its single-nucleotide polymorphism (SNP) rs143793106 in AgP by functional analyses of LIPA and its SNP in human periodontal ligament (HPDL) cells. GWAS was performed using the genome database of Japanese AgP patients, and the GWAS result was confirmed using Sanger sequencing. We examined the mRNA expression level of LIPA and the protein expression level of the encoded protein lysosomal acid lipase (LAL) in periodontium-composing cells using conventional and real-time polymerase chain reaction (PCR) and western blotting, respectively. Lentiviral vectors expressing LIPA wild-type (LIPA WT) and LIPA SNP rs143793106 (LIPA mut) were transfected into HPDL cells. Western blotting was performed to confirm the transfection. LAL activity of transfected HPDL cells was determined using the lysosomal acid lipase activity assay. Transfected HPDL cells were cultured in mineralization medium. During the cytodifferentiation of transfected HPDL cells, mRNA expression of calcification-related genes, alkaline phosphatase (ALPase) activity and calcified nodule formation were assessed using real-time PCR, ALPase assay, and alizarin red staining, respectively. The GWAS study identified 11 AgP-related candidate genes, including LIPA SNP rs143793106. The minor allele frequency of LIPA SNP rs143793106 in AgP patients was higher than that in healthy subjects. LIPA mRNA and LAL protein were expressed in HPDL cells; furthermore, they upregulated the cytodifferentiation of HPDL cells. LAL activity was lower in LIPA SNP-transfected HPDL cells during cytodifferentiation than that in LIPA WT-transfected HPDL cells. In addition, ALPase activity, calcified nodule formation, and calcification-related gene expression levels were lower during cytodifferentiation in LIPA SNP-transfected HPDL cells than those in LIPA WT-transfected HPDL cells. LIPA, identified as an AgP-related gene in a Japanese population, is expressed in HPDL cells and is involved in regulating cytodifferentiation of HPDL cells. LIPA SNP rs143793106 suppressed cytodifferentiation of HPDL cells by decreasing LAL activity, thereby contributing to the development of AgP.

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

In Vitro Bioactivity and Antibacterial Activity of Strontium-, Magnesium-, and Zinc-Multidoped Hydroxyapatite Porous Coatings Applied via Atmospheric Plasma Spraying.

The beneficial effects of Sr- and Mg-doped hydroxyapatite (HAp) on osteoblast proliferation and bone regeneration have been investigated in the past, and the antibacterial ability of Zn ions is well known. However, HAp coatings doped with these three elements via thermal spraying have not yet been investigated. In this study, HAp powder was synthesized at different pH values (4, 6, 8, and 10) and calcined at different temperatures (200, 400, 600, 800, and 1000 °C) to obtain HAp with the highest purity. Subsequently, strontium-, magnesium-, and zinc-doped HAp powders were synthesized at the optimal pH value and calcination temperature. The HAp powder was then coated onto Ti disks using atmospheric plasma spraying (APS) or vapor-induced pore-forming atmospheric plasma spraying (VIPF-APS) techniques at different working currents (350, 400, and 450 A) and spraying distances (10 and 15 cm). X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy equipped with energy-dispersive spectroscopy were used for material characterization to determine the optimal parameters. With these optimal coating parameters, HAp, Zn-HAp, SrMg-HAp, and ZnSrMg-HAp powders were deposited onto the Ti disks using VIPF-APS and named HAp-Ti, Zn-HAp-Ti, SrMg-HAp-Ti, and ZnSrMg-HAp-Ti, respectively. The in vitro bioactivity of these four groups was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and alkaline phosphatase (ALPase) activity assay. Besides, the antibacterial activities against Prevotella nigrescens, Porphyromonas gingivalis, and Fusobacterium nucleatum were assessed. The results showed that the purity of HAp synthesized at pH 10 and 800 °C was 98.40%. A porous coating without cracks was obtained at a 10 cm spraying distance and 400 A working current using VIPF-APS. SrMg-HAp-Ti and ZnSrMg-HAp-Ti resulted in higher osteoblast proliferation and ALPase activity than the control. Moreover, both Zn-HAp-Ti and ZnSrMg-HAp-Ti exhibited antibacterial activity against the three bacteria. Therefore, ZnSrMg-HAp has potential as a coating for biomedical materials due to its ability to reduce bacterial infection and enhance osseointegration.

Effect of Phosphate Solubilizing Bacteria on Phosphatase Activities, Yield and Phosphorus Nutrition of Wheat in an Acidic Entisol

This research aims to determine the short-term prospects of phosphate solubilizing bacteria (PSB) application on growth and yield of wheat and fertility status of soil with special reference to phosphorus (P). Spectrophotometric analyses were carried out to determine acid phosphatase (APase) and alkaline phosphatase (ALPase) activities in plant roots and rhizosphere soil, P bioavailability at the salient growth stages and harvest of the wheat crop, and P nutrition at maturity. Results of the wheat field experiment conducted during winter 2014–15 showed that application of PSB significantly increased the root APase but a quite opposite trend of the APase activity between plant roots and rhizosphere soil was found. In the case of ALPase activity, the effect of PSB was more prominent in root than soil. Among the two enzymes (APase and ALPase), APase activity was constantly higher in root and soil, indicating its importance in P hydrolysis and plant nutrition under acidic and P deficient soil conditions. The combined application of Pfertilizer and PSB had a profound influence on soil available P. Considerable effect of PSB on yield could be noted, but no significant effect was observed for P nutrition. The research work clearly indicated the importance of integration of P-fertilizer and PSB for maintaining higher crop productivity and better soil fertility. The study also emphasizes the need to consider the determinants which modulate the effectiveness of PSB in P solubilization and plant growth promotion.

A novel bio-active adhesive monomer induces odontoblast differentiation: a comparative study.

To evaluate the in vitro effect of the novel adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate (4-MET), on the proliferation, mineralization and differentiation of odontoblast-like cells, comparing with 4-MET, calcium hydroxide (CH) and mineral trioxide aggregate (MTA). Rat odontoblast-like MDPC-23 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% foetal bovine serum. The powder of four tested materials (CMET, 4-MET, CH and MTA) was first dissolved in distilled water (dH2O) and then was diluted by DMEM to yield final concentrations. Solvent (dH2O) was used as a control. Cell viability was assessed using CCK-8 assay. Real-time RT-PCR was used to quantify the mRNA expression of odontogenic markers, cytokines and integrins. Mineralization inducing capacity was evaluated by alkaline phosphatase (ALPase) activity and alizarin red S staining. Statistical analyses were performed using one-way anova and post hoc Tukey's HSD test, with the significance level at 1%. Cell viability was significantly greater in the CMET- (83 to 828mmolL-1), CH- and MTA-treated (low concentrations) groups than that in the control group (P<0.01). Higher concentrations of each material decreased the viable cells to different extents (P<0.01). CMET treatment augmented the expression of several integrin subunits and exhibited the highest mRNA expression levels of odontogenic markers among all groups (P<0.01). CH and MTA treatment caused significantly greater upregulation of pro-inflammatory cytokines expression than the other groups (P<0.01). The calcific deposition of MDPC-23 cells was dose-dependently accelerated by the addition of CMET (P<0.01); the enhancement of mineralization was also found in the fresh prepared CH and MTA treatments. Besides, CMET showed consistency in mineralization induction after 8weeks storage. Exposure to SB202190, a specific p38 mitogen-activated protein kinases inhibitor, significantly decreased the ALPase activity as well as the mineral deposition which was enhanced by CMET treatment (P<0.01). The novel bio-active monomer had the lowest cytotoxicity among all groups and it induced the proliferation, mineralization and differentiation of odontoblast-like cells under appropriate concentrations. This adhesive monomer possesses excellent biocompatibility and hence exhibits great potential in dentine regeneration.

Different effects of total flavonoids from Arachniodes exilis on human umbilical cord mesenchymal stem cells in vitro.

Traditional Chinese medicines are used in promotion of fractured bone healing and bone diseases. Some studies reported total flavonoids from plant can be used as an auxiliary source of exogenous.Use different methods to identify and verify effects of total flavonoids from Arachniodes exilis (TFAE) on human umbilical cord mesenchymal stem cells (HUCMSCs) in vitro.Concentrations of 1 and 5 μg/mL TFAE significantly increased ALPase activity in HUCMSCs compared to the other concentrations at days 3 and 7 (P < .05). RT-PCR showed that expression levels of osteogenic genes (Col1a1, OPN, Runx2 and Osx) were remarkably enhanced in HUCMSCs following treatment with different concentrations of TFAE for 9 days compared with 0 μg/mL TFAE group (control). The results showed that concentration < 5 μg/mL of TFAE induced osteogenic differentiation in HUCMSCs Alizarin red staining assays revealed that both TFAE and S1191 was significantly decreased (7.80 ± 0.66) compared with the TFAE group (16.00 ± 0.97) (P < .01). ALPase activity on days 3 and 7 was relatively lower in HUCMSCs grown in media supplemented with both S1191 and TFAE than that of in TFAE group only. The results indicated that osteogenic markers (Col1a1, OPN, Runx2 and Osx) were significantly downregulated in the TFAE + S1191 group in comparison to the control group. The expressions of Col1a and OPN in the TFAE + S1191 group decreased significantly (P < .01) by Western blotting.TFAE promotes the odonto/osteogenic differentiation of human UCMSCs via activation of ER.

Megestrol acetate induced proliferation and differentiation of osteoblastic MC3T3-E1 cells: A drug repurposing approach

AimsDrug repurposing or repositioning i.e.; identifying new indications for existing drugs have recently accelerated the process of drug discovery and development. Megestrol acetate (1) is a well-known progestin. It is commonly used as an appetite stimulant, and also in the treatment of breast, and endometrial cancers. The aim of this study is to investigate the effect of megestrol acetate (1) in osteoblast differentiation, and to determine the possible mechanism involved in megestrol acetate (1) induced osteoblast differentiation. Main methodsCytotoxicity of different steroidal drugs was evaluated using MTT assay. Alkaline phosphatase (ALP) activity was also determined, and alizarin red S (ARS) staining was performed to measure extracellular mineralization. Osteogenic protein levels were determined using Western blot analysis. Key findingsResults of the current study indicated that the megestrol acetate (1) enhanced the proliferation and differentiation of osteoblast cells at 1, 0.2, and 0.04 µM. This stimulatory effect of the megestrol acetate (1) was more prominent at 0.2 µM for cell proliferation, while the maximum cell differentiation (ALPase activity, and calcification) was observed at 0.04 μM. Western blot analysis also showed that megestrol acetate (1) altered the expression of bone morphogenic protein-2 (BMP2), p38, and pJNK proteins. Hence, only moderate doses of MGA (1) can enhance osteoblast proliferation and differentiation. SignificanceOur results identified that megestrol acetate (1) could be a potential lead for further research towards bone fragility related disorders.

Cellular Response of Anodized Titanium Surface by Poly(Lactide-co-Glycolide)/Bone Morphogenic Protein-2.

The purpose of this study is to examine physical characteristics of and initial biological properties to anodized titanium treated with poly(d,l-lactide-co-glycolide) (PLG) mixed with recombinant human bone morphogenic protein-2 (rhBMP-2). Titanium specimens were prepared in groups of four as follows: group NC was anodized under 300V as control; group PC was anodized then dropped and dried with solution 0.02ml PLG; group D was anodized then dropped and dried with solution 0.02ml PLG/rhBMP-2 (3.75μg per disc); and group E was anodized then coated with 0.02ml PLG/rhBMP-2 (3.75μg per disc) by electrospray. Human osteoblastic-like sarcoma cells were cultured. Cell proliferation and alkaline phosphatase (ALP) activity test were carried out. Runx-2 gene was investigated by the reverse transcription-polymerase chain reaction. Immunofluorescence outcome of osteogenic proteins was observed. After 3days, there were significantly higher proliferations compared rhBMP-2 loaded titanium discs with rhBMP-2 unloaded discs. The ALPase activity on rhBMP-2 loaded titanium discs was significantly higher than in rhBMP-2 unloaded discs. The expression level of Runx2 mRNA presented the highest on the PLG/rhBMP-2-coated surface. PLG polymers mixed with rhBMP-2 might improve proliferation, differentiation and osteogenic protein formation of cells on the anodized titanium.

The in vitro effects of CCN2 on odontoblast-like cells

ObjectiveTo investigate the in vitro effects of CCN2 on odontoblast-like cells proliferation and differentiation. DesignMDPC-23 cells were cultured in DMEM supplemented with 5% FBS. CCN2 was either added to culture media or coated onto culture polystyrene, addition or coating of dH2O was served as control. In the addition group, CCN2 (100 ng/mL) was added into culture media. In the coating group, CCN2 at the concentration of 1000 ng/mL was employed. Cell proliferation was performed using CCK-8 assay. Cell differentiation and mineralization were analyzed by ALPase activity assay, real time RT-PCR and alizarin red staining. One-way ANOVA with post-hoc tukey HSD test was used for statistical analysis. ResultsMDPC-23 cells exhibited robust proliferative activity upon exposure to either soluble or immobilized CCN2. ALP activity of cells cultured on CCN2-modified surface was continuously strengthened from day six (0.831 ± 0.024 units/μg protein versus 0.563 ± 0.006 units/μg protein of control) till day eight (1.035 ± 0.139 units/μg protein versus 0.704 ± 0.061 units/μg protein of control). Gene expression of BSP, OCN and OPN were promoted by soluble CCN2 after 48 h exposure. Moreover, gene expression of BSP, OCN, OPN, ALP, COL1 A1, Runx-2, DSPP and DMP-1 was significantly enhanced by immobilized CCN2. Finally, mineralization of MDPC-23 cells was accelerated by both soluble and immobilized CCN2 to different extent. ConclusionsThe findings indicate that CCN2 promoted proliferation, odontogenic gene expression and mineralization of MDPC-23 cells. It is proposed that CCN2 may be a promising adjunctive formula for dentin regeneration.

Knockdown of NRAGE induces odontogenic differentiation by activating NF-κB signaling in mouse odontoblast-like cells

ABSTRACTPurpose: Neurotrophin receptor-interacting MAGE homologue (Nrage) plays an important role in bone development and the metabolism of normal skeletal structures. Our previous study showed that Nrage inhibited the odontogenic differentiation of mouse dental pulp cells. However, the potential roles and mechanism of Nrage in regulating odontogenic differentiation are unknown. The aim of this study was to investigate the molecular mechanism of Nrage in odontogenic differentiation of mouse odontoblast-like cells.Materials and methods: Endogenous expression of Nrage was stably downregulated by lentivirus-mediated shRNA. Mineralized nodules formation was detected by alizarin red S staining. Dmp-1, Dspp, and ALP mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. In addition, ALPase activity was detected. Confocal microscopy and co-immunoprecipitation (co-IP) were used to analyze the interactions between NRAGE and NF-κB signaling molecules. An IKK inhibitor was also used in the study.Results: NRAGE expression in odontoblasts was downregulated during mouse first maxillary molar development. Moreover, NRAGE expression was downregulated during odontogenic differentiation of odontoblast-like cells. NRAGE knockdown significantly upregulated DMP1 and DSP expression, increased ALPase activity, and promoted mineralized nodule formation. In addition, NRAGE knockdown increased the translocation of NF-κB1 to the nucleus and phosphorylation levels of p65. Co-IP results showed that NRAGE bound to IKKβ. Most importantly, the promoting effect of Nrage knockdown on odontoblastic differentiation was reduced after treatment with an IKK inhibitor.Conclusions: Our data confirmed that NRAGE is an important regulator of odontogenic differentiation of odontoblasts by inhibiting the NF-κB signaling pathway through binding to IKKβ.Abbreviations: Nrage: neurotrophin receptor-interacting MAGE homologue; DSP: dentin sialophospho protein; DMP-1: dentin matrix protein-1; BMP: bone morphogenetic protein; Wnt: wingless; NF-κB: nuclear factor of activated B cells; DAPI: 4′,6-diamidino-2-phenylindole; KO: knockout; DPCs: dental pulp cells; AA: ascorbic acid; β-Gly: β-glycerophosphate; Dex: dexamethasone; co-IP: co-immunoprecipitation; IκB: inhibitor of NF-κB; IKK: IκB kinase

FAM20C could be targeted by TET1 to promote odontoblastic differentiation potential of human dental pulp cells.

Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation. The expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter. FAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction. TET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression.

Effects of paraoxonase 1 on the cytodifferentiation and mineralization of periodontal ligament cells.

Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P=.0190). PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.

Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts.

ObjectiveClinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/β-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. In addition to the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. However, there are no reports of either pathway in OA Ob. Here, we studied the two non-canonical pathways in OA Ob and if they influence their phenotype.MethodsHuman primary subchondral Ob were isolated from the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or of normal individuals at autopsy. The expression of genes involved in non-canonical Wnt signaling was evaluated by qRT-PCR and their protein production by Western blot analysis. Alkaline phosphatase activity and osteocalcin secretion (OC) were determined with substrate hydrolysis and EIA, respectively. Mineralization levels were evaluated with Alizarin Red Staining, Wnt/PKC and Wnt/PCP pathways by target gene expression and their respective activity using the NFAT and AP-1 luciferase reporter assays.ResultsOA Ob showed an altered phenotype as illustrated by an increased alkaline phosphatase activity and osteocalcin release compared to normal Ob. The expression of the non-canonical Wnt5a ligand was increased in OA Ob compared to normal. Whereas, the expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar. Wnt5a directly stimulated the expression and production of LGR5, contrasting, Wnt5a did not stimulate the expression of LGR4. Wnt5a also stimulated the phosphorylation of both JNK and PKC, as well as the activity of both NFAT and AP-1 transcription factors. The inhibition of Wnt5a expression partially corrects the abnormal mineralization, OC secretion and ALPase activity of OA Ob.ConclusionThese data indicate that the alteration of Wnt5a, a non-canonical Wnt signaling activator, is implicated in the modified signalisation and phenotype observed in OA Ob.

Biocompatibility of Titanium Carbo-Nitride Coatings with Fetal Rat Calvarial Cells

Surface modification is a promising technique to improve mechanical properties of titanium and titanium alloys by changing the surface composition or microstructures. In this study, titanium carbo-nitride coating had been deposited on Ti-6Al-4V alloy by arc ion plating. The biocompatibility of TiN and TiCN coating on Ti-6Al-4V alloy with fetal rat calvarial cells was assessed by Enzyme Linked Immunoabsorbent Assay (ELISA) and Alkaline phosphatase (ALPase) activity. The effect of surface coating on IL-1β and IL-6 production was determined by ELISA. On IL-1β and IL-6 production, there were no statistical difference between control and thin coating surfaces. After 7 days of culture, the cells were provided to measure ALPase activity. ALPase activity of cells cultured on TiN and TiCN surface was similar to that seen on cp-Ti.

Osteogenic potency of dedifferentiated fat cells isolated from elderly people with osteoporosis.

Mature adipocytes are the major cell type in adipose tissue. This study aimed to explore the osteogenic potency of dedifferentiated fat cells obtained from osteoporotic patients (opDFATs) in vitro and in vivo. Mature adipocytes and adipose-derived stem cells (opASCs) were harvested from subcutaneous adipose tissue. Mature adipocytes were dedifferentiated to produce opDFATs by the ceiling culture method. OpDFATs were osteogenically induced in vitro with opASCs as a control. Cell growth, alkaline phosphatase (ALPase) activity and cell mineralization were determined, and expression levels of osteogenesis-specific genes (collagen I, osteocalcin and bone sialoprotein) were analyzed using quantitative reverse transcription polymerase chain reaction. After 14 days, the opDFATs were combined with a poly(lactide-co-glycolide)-β-tricalcium phosphate porous scaffold after being suspended in collagen I gel and implanted into nude mice for 4 weeks prior to histological analysis. Unilocular lipid droplets in mature adipocytes gradually split into smaller droplets and disappeared from the cytoplasm. Mature adipocytes dedifferentiated to opDFATs and cell morphology changed from spherical to elongated. High levels of ALPase and cell mineralization were observed in opDFATs by staining. No significant differences were found between the growth curves, ALPase activity, cell mineralization and expression levels of osteogenesis-specific genes between opDFATS and opASCs. After implantation for 4 weeks, new bone tissue was observed histologically in the opDFATs-based biocomposite. OpDFATs are implicated as a novel type of seed cell for bone tissue engineering based on their osteogenic potency and higher abundance in adipose tissue compared with opASCs.

Osteoconductivity of Binary Titanium Alloys with Different Micro/Nanoporous Surfaces.

The surface characteristics and osteoconductivity were evaluated for the micro/nanoporous surfaces of titanium (Ti) alloys prepared by micro-arc oxidation (MAO) and hydrothermal treatment (HT) of binary Ti-5 wt% A alloys (A = Au, Mn, Nb, and Pd). Surface properties were analyzed using X-ray diffractometry, scanning electron microscopy, and energy dispersive X-ray spectroscopy. The osteoconductivity was evaluated by measuring the total protein, ALPase activity, and osteocalcin production. The surface morphologies of MAO/HT specimens mainly affected on their osteoconductivity. Total proteins on Ti alloys (MAO/HT) were slightly lower than that on commercially pure Ti (MAO/HT) after incubation of MG-63 osteoblast-like cells for 14 days. However, better ALPase activity and osteocalcin production were observed on MAO/HT-treated Ti–5Mn, Ti–5Nb, and Ti–5Pd than that on cp-Ti (MAO/HT) after 14 days. Especially, Ti–5Mn (MAO/HT) showed a significant increase of ALPase activity due to its well grown micro/nano structure. Meanwhile, very small nanorods on Ti–5Au (MAO/HT) affected negatively to ALPase activity and osteocalcin production.

The effect of delta-like 1 homologue on the proliferation and odontoblastic differentiation in human dental pulp stem cells.

This study aimed to investigate the functions of delta-like homologue 1 (DLK1) in the proliferation and differentiation of human dental pulp stem cells (hDPSCs). Immunohistochemical analysis was used to determine the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), DLK1, NOTCH1 and p-ERK1/2 in the mouse first maxillary molar. Recombinant lentivirus was constructed to overexpress DLK1 stably in hDPSCs. The cell viability and proliferation of hDPSCs were examined by CCK8 and EdU incorporation assay respectively. The odontoblastic differentiation of hDPSCs was determined by detection of ALPase activity assay, ALP and alizarin red staining and the expression of mineralization-related genes including ALP, DSPP and dental matrix protein. The mRNA and protein levels of DLK1 and p-ERK1/2 protein expression were detected. ERK inhibitor was used to test the differentiation effect of DLK1 on hDPSCs. Delta-like homologue 1 was highly expressed on the odontoblasts and dental pulp cells on the first maxillary molar; the expression of p-ERK1/2 is similar with the DLK1 in the same process. The expression level of DLK1 increased significantly after the odontoblastic induction of hDPSCs. DLK1 overexpression increased the proliferation ability of hDPSCs and inhibited odontoblastic differentiation of hDPSCs. The protein level of p-ERK1/2 significantly increased in hDPSCs/dlk1-oe group. ERK signalling pathway inhibitor reversed the odontoblastic differentiation effects of DLK1 on hDPSCs. The proliferation of hDPSCs was promoted after DLK1 overexpression. DLK1 inhibited the odontoblastic differentiation of hDPSCs, which maybe through ERK signalling pathway.

Tumor Proteins D52 and D54 Have Opposite Effects on the Terminal Differentiation of Chondrocytes.

The tumor protein D (TPD) family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase) activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.