Phosphatase mRNA Research Articles

Long non-coding TRPM2-AS regulates fracture healing by targeting miR-545-3p/Bmp2

ObjectiveDelayed fracture healing increases the suffering of patients. An in-depth investigation of the pathogenesis of delayed fracture healing may offer new direction for the prevention and treatment.MethodsThe study included 63 normal healing tibial fractures and 58 delayed healing tibial fractures patients. Long non-coding RNA (lncRNA)TRPM2-AS, microRNA-545-3p (miR-545-3p), bone morphogenetic protein 2 (Bmp2) mRNA and osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and alkaline phosphatase (Alp) mRNA expression were determined by Real-time quantitative reverse transcription-polymerase chain reaction in serum and MC3T3-E1 cells. The prediction potential of TRPM2-AS in delayed healing fracture patients was verified by receiver operating characteristic curves. The binding relationship of TRPM2-AS/miR-545-3p/Bmp2 was evaluated by dual luciferase reporter gene assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.ResultsTRPM2-AS was remarkably down-regulated in patients with delayed fracture healing and could better predict the fracture healing status. TRPM2-AS downregulation inhibited osteogenic markers mRNA expression, restrained proliferation, and promoted apoptosis of MC3T3-E1 cells (p < 0.05). In delayed fracture healing, miR-545-3p was dramatically up-regulated and was negatively regulated by TRPM2-AS. Reducing miR-545-3p eliminate the negative effect of TRPM2-AS down-regulation on osteoblast proliferation and differentiation (p < 0.05). miR-545-3p targets Bmp2, which plays a positive role in osteoblast differentiation (p < 0.05).ConclusionThis study found that TRPM2-AS has the potential to be a diagnostic marker for delayed fracture healing and revealed that the TRPM2-AS/miR-545-3p/Bmp2 axis affects fracture healing by regulating osteoblast.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 μM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity.

Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolated from these sources and expanded through two passages. Basal expression of Runx2 and Osterix was assessed in undifferentiated third passage cells, along with their response to osteogenic culture conditions. Bone marrow-derived cells had significantly higher basal expression of Osterix, but not Runx2. In osteogenic medium, bone-marrow cells rapidly developed dense, multicellular aggregates that stained strongly for mineral and alkaline phosphatase activity. Synovial and adipose cell cultures showed far less matrix mineralization. Bone marrow cells significantly up-regulated alkaline phosphatase mRNA expression and enzymatic activity at 7 and 14 days. Alkaline phosphatase expression and activity were increased in adipose cultures after 14 days, although these values were less than in bone marrow cultures. There was no change in alkaline phosphatase in synovial cultures. In osteogenic medium, bone marrow cultures increased both Runx2 and Osterix mRNA expression significantly at 7 and 14 days. Expression of both transcription factors did not change in synovial or adipose cultures. These results demonstrate that basal Osterix expression differs significantly in progenitor cells derived from different tissue sources and reflects the osteogenic potential of the cell populations.

Influence of a Physiologically Formed Blood Clot on Pre-Osteoblastic Cells Grown on a BMP-7-Coated Nanoporous Titanium Surface.

Titanium (Ti) nanotopography modulates the osteogenic response to exogenous bone morphogenetic protein 7 (BMP-7) in vitro, supporting enhanced alkaline phosphatase mRNA expression and activity, as well as higher osteopontin (OPN) mRNA and protein levels. As the biological effects of OPN protein are modulated by its proteolytic cleavage by serum proteases, this in vitro study evaluated the effects on osteogenic cells in the presence of a physiological blood clot previously formed on a BMP-7-coated nanostructured Ti surface obtained by chemical etching (Nano-Ti). Pre-osteoblastic MC3T3-E1 cells were cultured during 5 days on recombinant mouse (rm) BMP-7-coated Nano-Ti after it was implanted in adult female C57BI/6 mouse dorsal dermal tissue for 18 h. Nano-Ti without blood clot or with blood clot at time 0 were used as the controls. The presence of blood clots tended to inhibit the expression of key osteoblast markers, except for Opn, and rmBMP-7 functionalization resulted in a tendency towards relatively greater osteoblastic differentiation, which was corroborated by runt-related transcription factor 2 (RUNX2) amounts. Undetectable levels of OPN and phosphorylated suppressor of mothers against decapentaplegic (SMAD) 1/5/9 were noted in these groups, and the cleaved form of OPN was only detected in the blood clot immediately prior to cell plating. In conclusion, the strategy to mimic in vitro the initial interfacial in vivo events by forming a blood clot on a Ti nanoporous surface resulted in the inhibition of pre-osteoblastic differentiation, which was minimally reverted with an rmBMP-7 coating.

Oxidative stress impairs the calcification ability of human dental pulp cells

BackgroundThe relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H2O2) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3).Material and methodshDPCs (Lonza, Basel, Switzerland) were exposed to H2O2. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H2O2 on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor.ResultsH2O2 at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H2O2-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H2O2-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD.ConclusionTo the best of our knowledge, this is the first study documenting the involvement of IP3 signaling in the calcification ability of human dental pulp cells impaired by H2O2.

Strontium Regulates the Proliferation and Differentiation of Isolated Primary Bovine Chondrocytes via the TGFβ/SMAD Pathway.

The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor β (TGFβ) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 μg/ml, as SrCl2·6H2O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 μM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 μg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFβ1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFβ1/ALK5-induced SMAD3 phosphorylation, and at 10 μg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 μg/ml. In contrast, Sr at 10 μg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 μg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 μg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFβ1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.

Short-chain fatty acids, acetate and propionate, directly upregulate osteoblastic differentiation

Short-chain fatty acids, including acetate, propionate, and butyrate are metabolites of dietary fibre produced by microbiota in the large intestine, have been proposed to contribute to effects on bone homeostasis. However, it is unclear whether they are used in osteoblasts and directly affect bone formation. We investigated whether short-chain fatty acids are absorbed in osteoblast cells and influence early osteoblastic differentiation using MC3T3-E1 cells. Acetate and propionate upregulated alkaline phosphatase activity, which is an osteoblast differentiation marker, and acetate upregulated alkaline phosphatase mRNA expression after treatment for 9 days, whereas butyrate did not in MC3T3-E1 cells. Butyrate was absorbed more rapidly and to a greater extent than acetate and propionate. These results indicate that short-chain fatty acids were used in osteoblastic cells, and particularly acetate and propionate directly upregulated differentiation in primary osteoblasts. Therefore, acetate and propionate might be useful for maintaining a positive balance of bone turnover.

Evaluation of optimal dietary calcium level by bone characteristics and calcium metabolism-related gene expression of broilers from 22 to 42 d of age.

The current dietary Ca recommendation of broilers is primarily based on the previous studies carried out more than 30 yr ago. However, the modern commercial broilers are quite different from those more than 30 yr ago. The present experiment was conducted to evaluate an optimal dietary Ca level by bone characteristics and Ca metabolism-related gene expression of broilers fed a corn-soybean meal diet from 22 to 42 d of age. A total of 252 22-d-old Arbor Acres male broilers were randomly assigned to 1 of 7 treatments with 6 replicate cages of 6 birds per cage for each treatment. Broilers were fed the corn-soybean meal diets containing 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, or 1.10% Ca for 21 d, and each diet contained 0.31% non-phytate P. The results showed that the mineral contents in tibia and middle toe bone, mineral density in tibia and middle toe bone, middle toe ash percentage, middle toe ash Ca percentage, and tibia alkaline phosphatase mRNA expression level of broilers were influenced (P < 0.04) by dietary Ca level and increased quadratically (P < 0.05) as dietary Ca level increased. The estimates of optimal dietary Ca levels were 0.55%, 0.60%, 0.70%, 0.72%, 0.63%, 0.66%, and 0.70%, respectively, based on the best fitted broken-line, quadratic, or asymptotic models (P < 0.02) of the above sensitive indices. These results indicate that the optimal dietary Ca level would be 0.72% to support all of the Ca metabolism and bone development of broilers fed the corn-soybean meal diet from 22 to 42 d of age.

The relationship of telmisartan with sclerostin in the osteoporosis model induced by ovariectomy in rats.

Our aim is to explain the relationship between Ang II and Scl in osteoporotic (OP) rats and the contribution of Scl in the antiosteoporotic effect mechanism of angiotensin receptor blockers (ARB). This study consists of two sub-studies conducted on 4th and 12th weeks after ovariectomy. In study 1, treatment was started immediately after bilateral ovariectomy (OVX), while, in study 2, treatment was started 2 months after OVX. Two different doses of telmisartan (5 and 10 mg/kg) were administered with the aid of gavage for 30 days in both sub-study groups. Serum and tissue Scl, osteocalcin, osteopontin and tartrate-resistant acid phosphatase mRNA expressions were higher and bone mineral densities (BMD) and bone-specific alkaline phosphatase (BALP) mRNA expressions were found to be lower in the OVX groups compared with the sham group. In OVX groups where two different doses of telmisartan were administered, BMD and BALP mRNA expressions increased and serum and tissue Scl decreased. There may be a close relationship between angiotensin II and sclerostin in the development of osteoporosis. In this study, telmisartan administration showed an antiosteoporotic effect and significantly decreased the level of sclerostin. These results strongly support this relationship.

Naringin Improves Osteoblast Mineralization and Bone Healing and Strength through Regulating Estrogen Receptor Alpha-Dependent Alkaline Phosphatase Gene Expression.

Phytoestrogens are strongly recommended for treating osteoporosis. Our previous study showed that naringin, a citrus flavonoid, can enhance the bone mass in ovariectomized rats. In this study, we further elucidated the mechanisms of naringin-induced osteoblast maturation and bone healing. Treatment of human osteoblasts with naringin increased cell viability and proliferation. In parallel, exposure to naringin enhanced translocation of estrogen receptor alpha (ERα) to nuclei and its transactivation activity. Sequentially, naringin induced alkaline phosphatase (ALP) mRNA and protein expression and its enzyme activity. Pretreatment with methylpiperidinopyrazole (MPP), a specific inhibitor of ERα, attenuated naringin-induced augmentations in ERα transactivation activity, ALP gene expression, and cell mineralization. The beneficial effects of naringin were also confirmed in mouse MC3T3-E1 cells. Moreover, administration of mice with a bone defect with naringin increased levels of ERα and ALP in damaged sites and simultaneously enhanced the healing rate and bone strength. Nevertheless, treatment with MPP weakened naringin-triggered expression of ERα and ALP and improved bone healing and mass. Therefore, naringin could improve osteoblast mineralization and bone healing via regulating ERα-dependent ALP gene expression. Naringin can be clinically applied for treatment of osteoporosis-related bone diseases.

A study to explore the effects of low dietary protein levels on the growth performance and nutritional metabolism of grass carp (Ctenopharyngodon idella) fry

An 8-week feeding trial was conducted to investigate the effects of low dietary protein levels (DPLs) on the growth performance and nutritional metabolism of grass carp (Ctenopharyngodon idella) fry. The results showed that the average final weight (AFW), weight gain rate (WGR) and specific growth rate (SGR) reached the maximum values at 43.30% DPL, and then became constant (P < 0.05). The feed conversion ratio (FCR) showed an opposite trend (P < 0.05). Low DPLs (18.20% and 24.30%) suppressed the survival rate (SR) (P < 0.05). The protein efficiency ratio (PER) reached the maximum value at 36.40% DPL (P < 0.05). The moisture content was lowered by at 36.40% DPL (P < 0.05). The crude protein content was improved by 43.30% DPL (P < 0.05). Low DPLs (18.20% and 24.30%) significantly increased the fat composition compared to other DPLs (P < 0.05). 43.30% DPL significantly promoted the rapamycin target protein (TOR) and ribosomal protein S6 kinase-polypeptide 1 (S6K1) mRNA levels, but inhibited the 4E binding protein 1 (4EBP1) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) mRNA levels (P < 0.05). Low DPLs (18.20% and 24.30%) increased fat synthesis-related genes, the mRNA levels of lipolysis-related genes were significantly higher at 43.30% DPL (P < 0.05). The glucokinase (GK) mRNA levels at 18.20%, 24.30% and 30.60% DPLs were higher (P < 0.05). The glucose 6 phosphatase (G6Pase) mRNA level was higher at 43.30% DPL (P < 0.05). Hepatopancreas transcriptome results showed that compared with the low DPL (18.20%), the appropriate DPL (43.30%) significantly affected the amino acid metabolism and lipid metabolism. This study therefore, demonstrated that low DPLs adversely affected the growth and nutrient metabolism in grass carp fry.

Pulsed-electromagnetic-field induced osteoblast differentiation requires activation of genes downstream of adenosine receptors A2A and A3.

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

Periodontal ligament cells in adolescents and adults: Genetic level responses to orthodontic forces

Periodontal ligament cells (PDLC) respond to the application of mechanical forces by releasing various molecules that participate in bone remodeling. Whether these cellular reactions take place at the same rate in adolescent and adult patients is not known. In this study, we aimed to evaluate differences in genetic expression, if any, between the release of various inflammatory mediators from PDLC in adolescent and adult patients before and after the application of orthodontic forces. Forty subjects with bimaxillary dentoalveolar protrusion requiring extraction of first premolars for orthodontic treatment were selected and divided into 2 groups. Group A included 20 adolescents (aged 12-20years), and group B included 20 adults (aged 35-50years). Then, 35-50g of force were applied to the maxillary first premolars, and teeth were extracted at different periods: pretreatment (control group), 7days, 14days, and 28days (experimental group). The periodontal ligament was scraped from the middle third of the root, and the beta-galactosidase assay was performed in the control group. RNA extraction, DNase treatment, quantitative polymerase chain reaction, and complementary DNA synthesis were performed in the experimental group. Adult PDLC exhibited senescent changes through increased beta-galactosidase activity. The increase in the inflammatory response and bone resorption in adult patients was evident by increased prostaglandin E2, IL1B, and acid phosphatase mRNA expression levels. Controlled bone formation response by adolescent PDLC was evident from increased ALP and BGLAP mRNA levels and a balanced receptor activator of nuclear factor kappa-Β ligand/OPG ratio. The study could identify the reasons behind the differential response of adolescent and adult PDLC to orthodontic mechanics.

Examining the levels of acetylation, DNA methylation and phosphorylation in HIV-1 positive and multidrug-resistant TB-HIV patients

ObjectivesIn this study, we examined the impact of epigenetic modifications on host gene functioning by assessing the expression of seven candidate genes in three separate groups including healthy, multidrug-resistant (MDR) TB-HIV co-infected and HIV-1 positive individuals. MethodsTen patients with MDR TB and HIV-1 co-infection on TB and HIV therapy and a cohort comprised of 10 newly diagnosed individuals with HIV-1 infection were recruited from the TB and HIV clinics at the Charlotte Maxeke Johannesburg Academic Hospital. Notably, the HIV-1 positive individuals were not placed on antiretroviral therapy (ART) at the time of recruitment and blood collection. A third group consisting of 10 healthy participants without MDR TB or HIV infection was recruited from the University of the Witwatersrand. Blood samples collected from all three cohorts were employed for extraction of plasma, total RNA and genomic DNA. ResultsOur data indicated that the expression of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) genes was significantly increased in HIV-1 positive patients and was lowest in MDR TB-HIV co-infected patients. By contrast, histone acetyltransferase (HAT), histone deacetylase (HDAC), protein tyrosine kinase (PtkA) and protein tyrosine phosphatase (PtpA) mRNA expression levels were substantially enhanced in HIV-1 infected and were lowest in healthy individuals. Conversely, Dicer expression levels were comparable among all three study groups. ConclusionPromising preliminary data emanating from this investigation may potentially be used for generation of novel vaccines and therapeutic compounds capable of neutralising MDR TB-HIV and HIV-1 infection.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. Invivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Synthesis and Characterization of Melatonin-Loaded Chitosan Microparticles Promote Differentiation and Mineralization in Preosteoblastic Cells.

In terms of a novel scaffold with well good osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation. The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro. The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy. The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on preosteoblastic MC3T3-E1 cells was characterized in vitro. An in vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period. In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced alkaline phosphatase (ALP) mRNA expression and ALP activity compared with the control group. Meanwhile, the osteoblast-specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), collagen I (Col I), and osteocalcin (Ocn), were also significantly upregulated. Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared with controls. In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of preosteoblast cells in vitro.

Tissue alkaline phosphatase activity and expression in an experimental infant swine model of cardiopulmonary bypass with deep hypothermic circulatory arrest

BackgroundInfant cardiac surgery with cardiopulmonary bypass results in decreased circulating alkaline phosphatase that is associated with poor postoperative outcomes. Bovine intestinal alkaline phosphatase infusion represents a novel therapy for post-cardiac surgery organ injury. However, the effects of cardiopulmonary bypass and bovine-intestinal alkaline phosphatase infusion on tissue-level alkaline phosphatase activity/expression are unknown.MethodsInfant pigs (n = 20) underwent cardiopulmonary bypass with deep hypothermic circulatory arrest followed by four hours of intensive care. Seven control animals underwent mechanical ventilation only. Cardiopulmonary bypass/deep hypothermic circulatory arrest animals were given escalating doses of bovine intestinal alkaline phosphatase infusion (0-25 U/kg/hr.; n = 5/dose). Kidney, liver, ileum, jejunum, colon, heart and lung were collected for measurement of tissue alkaline phosphatase activity and mRNA.ResultsTissue alkaline phosphatase activity varied significantly across organs with the highest levels found in the kidney and small intestine. Cardiopulmonary bypass with deep hypothermic circulatory arrest resulted in decreased kidney alkaline phosphatase activity and increased lung alkaline phosphatase activity, with no significant changes in the other organs. Alkaline phosphatase mRNA expression was increased in both the lung and the ileum. The highest dose of bovine intestinal alkaline phosphatase resulted in increased kidney and liver tissue alkaline phosphatase activity.ConclusionsChanges in alkaline phosphatase activity after cardiopulmonary bypass with deep hypothermic circulatory arrest and bovine intestinal alkaline phosphatase delivery are tissue specific. Kidneys, lung, and ileal alkaline phosphatase appear most affected by cardiopulmonary bypass with deep hypothermic circulatory arrest and further research is warranted to determine the mechanism and biologic importance of these changes.

In Vitro and In Vivo Osteogenesis Induced by Icariin and Bone Morphogenetic Protein-2: A Dynamic Observation.

In the present study, we aimed to compare the effects of icariin (ICA) and bone morphogenetic protein-2 (BMP-2) on osteoblast proliferation and osteogenesis in bone defects. We found that in vitro ICA or BMP-2 treatment is able to increase osteoblast proliferation, which was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Specifically, ICA at a concentration of 30 µg/ml had the strongest ability to promote cell proliferation, which is equivalent with the effect of BMP-2 at a concentration of 50 µg/ml. Furthermore, Western blot and RT-qPCR analyses showed that treatment with ICA (20–30 µg/ml) had similar increase effect with BMP-2 (50 µg/ml) on the protein and mRNA levels of BMP-2, osteoprotegerin (OPG), and alkaline phosphatase (ALP) mRNAs. In addition, the animal model of bone defects was successfully prepared. The in vivo data showed that compared with the control group, highest osteogenesis in the ICA or BMP-2 groups was observed at different observational times. Four weeks after surgery, osteogenesis in the BMP-2 group was slightly higher than that in the ICA group, but there was no significant difference between the two groups until the eighth week. ICA promotes osteoblast proliferation by stimulating the expression of BMP-2 and OPG proteins and upregulating the expression of BMP-2, OPG, and ALP mRNAs. ICA at a certain concentration has the same osteogenic effect as BMP-2. ICA or BMP-2 composite nanomaterials can be used as a framework to guide bone regeneration and promote osteogenesis. In addition, the combined use of hematoxylin-eosin and Goldner’s trichrome staining techniques contributes to acquiring better bone morphometric information about bone defects.

Screen and Function Study of the Key Regulative Odontogenic Genes in Mice

To screen the key odontogenic genes in mice and verify the odontogenic inducing effect on amniotic epithelial cells (WISH). The spatially and temporally different expression of bone morphogenetic proteins 4 (BMP4), fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), lymphoid enhancer factor 1 (LEF1) proteins and their genes expression in the early odontogenesis stage (embryo day 10.5 (E10.5)、E11.5、E14.5) in fetal mice were detected by immunohistochemistry staining and quantitative real-time PCR (RT-qPCR). According to the results, we screened the probable key odontogenic genes. Then adding osteogenic inducing solution to induce non-odontogenic epithelium cells, WISH. After 3 weeks culture of non-odontogenic epithelial WISH for osteogenic induction, the epithelial-mesenchymal transformation cap ability was evaluated by using Alizarin (ALZ) red staining and RT-qPCR on the alkaline phosphatase ( ALP) mRNA expression level. Using germ layer recombination experiment to observe and verify whether the screened genes can induce non-odontogenic epithelium cells acquire odontogenesis ability. The recombined tissue grafts containing key genes were transplanted beneath the renal capsule of mice. The results of immunohistochemistry staining and RT-qPCR showed that on E10.5 BMP4 protein and gene were differently expressed in the first and second branchial arch epithelium, which synchronized the odontogenic capability transferring from epithelium to mesenchyme from E10.5-E14.5. Though the expression of FGF8 protein and gene existed such difference in the first and second branchial arch epithelium, there was no synchronization in transfer. The expression of LEF1 and SHH proteins and genes had neither difference nor synchronization. So far, we considered the BMP4 was the probable key odontogenic gene. Through 3 weeks' osteogenic induction, ALZ red stained positively and calcium nodules were observed in WISH, and the expression level of ALP mRNA increased. In the germ layer recombination experiment, exogenous BMP4 protein enabled the second branchial arch mesenchyme forming tooth-like structures after recombined with the second branchial arch epithelium or WISH. The proteins and genes of BMP4, FGF8, SHH and LEF1 are spatially and temporally differently expressed in the early tooth development stage in mice. The protein and gene of BMP4 are differently expressed between the first and second branchial arch epithelium and enables the non-odontogenic epithelium acquiring odontogenic ability. BMP4 is the possible key odontogenic gene.

251. Additive manufactured Ti-6Al-4V/polyetheretherketone composite porous cage for interbody fusion: bone growth and biocompatibility evaluation in a porcine model

BACKGROUND CONTEXT Several materials have been used to manufacture the cages, the most common being polyetheretherketone (PEEK) and titanium (Ti) alloy (Ti–6Al–4V). Each material has its advantages and disadvantages. PURPOSE The purpose is to utilize the advantages of each material in combination with additive manufacturing technology to develop a new porous Ti alloy/PEEK composite interbody cage. STUDY DESIGN/SETTING In vitro mechanical tests and cell biocompatibility study; in vivo posterior instrumented interbody fusion porcine model. PATIENT SAMPLE In vitro cell culture analysis for Porous Ti alloy/PEEK composite cages. In vivo animal study used 20 female pigs through an anterior intervertebral lumbar fusion and posterior pedicle screws augmentation. Each level was randomly implanted with one of five testing cages. OUTCOME MEASURES In vitro mechanical tests for shear strength of the interface layer. In vitro cell culture analysis for alkaline phosphatase (ALP) activity, ALP and osteocalcin mRNA expression for in vitro study. Micro–computed tomography (CT), back-scattered-electrons SEM (BSE-SEM) and histological analyses for in vivo study. METHODS In vitro and in vivo study compared 5 groups of cages. The first was a commercialized pure PEEK cage (group 1). The second was a Ti alloy/PEEK composite cage with nonporous Ti alloy end plates (group 2). The third, fourth, and fifth groups were composite cages with porosity of 40%, 60%, and 80% (groups 3, 4, and 5), respectively. Mechanical tests, alkaline phosphatase activity and mRNA expression, osteocalcin mRNA expression, SEM were performed for in vitro study. Micro–computed tomography (CT), back-scattered-electrons SEM (BSE-SEM) and histological analyses were performed for in vivo study. RESULTS The shear strength reached 33.4 MPa at the Ti alloy/PEEK interface. ALP activity and ALP mRNA expression were positively correlated with the porosity rate and peaked at 60% porosity. Micro-CT and BSE-SEM demonstrated that structures with high porosities, especially 60% and 80%, facilitated more bone formation inside the implant but not outside the implants. Histological analysis also showed that bone formation was better in Ti alloy groups than in the PEEK group. CONCLUSIONS In vitro cell culture showed higher porous structure exhibited superior bone growth. Micro-CT and histological analyses showed improved bone growth in high-porosity composite cage groups. Composite cage implant takes the biological advantages of Ti alloy porous end plates and the mechanical and radiographic advantages of PEEK central core to incorporate as a single implant suitable for intervertebral fusion. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.