Alkaline Phosphatase Gene Expression Research Articles

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach

IntroductionHuman dental pulp cells (HDPCs) are generally isolated and cultured with xenogeneic products and in stress conditions that may alter their biological features. However, guidelines from the American Food and Drug Administration and the European Medicines Agency currently recommend the use of protocols compliant with medicinal manufacturing. Our aim was to design an ex vivo procedure to produce large amounts of HDPCs for dentin/pulp and bone engineering according to these international recommendations. MethodsHDPC isolation was performed from pulp explant cultures. After appropriate serum-free medium selection, cultured HDPCs were immunophenotyped with flow cytometry. Samples were then cryopreserved for 510 days. The post-thaw cell doubling time was determined up to passage 4 (P4). Karyotyping was performed by G-band analysis. Osteo/odontoblastic differentiation capability was determined after culture in a differentiation medium by gene expression analysis of osteo/odontoblast markers and mineralization quantification. ResultsImmunophenotyping of cultured HDPCs revealed a mesenchymal profile of the cells, some of which also expressed the stem/progenitor cell markers CD271, Stro-1, CD146, or MSCA-1. The post-thaw cell doubling times were stable and similar to fresh HDPCs. Cells displayed no karyotype abnormality. Alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein gene expression and culture mineralization were increased in post-thaw HDPC cultures performed in differentiation medium compared with cultures in control medium. ConclusionsWe successfully isolated, cryopreserved, and amplified human dental pulp cells with a medicinal manufacturing approach. These findings may constitute a basis on which to investigate how HDPC production can be optimized for human pulp/dentin and bone tissue engineering.

Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression.

Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2h and enhanced filopodia growth at 0.5h. Significantly, more osteoblasts were also observed on TNT/FBS after 7d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3d and 90% at 5d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7d and 14d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression.

Characterization of progenitor cells and stem cells from the periodontal ligament tissue derived from a single person.

Periodontal ligament progenitor cells (PDLPs) and PDL stem cells (PDLSCs) are progenitor and stem cells that were isolated from PDL tissues using the outgrowth and single cell isolation methods respectively. The differences between PDLPs and PDLSCs characteristics could be observed from previous studies. However, these cells were obtained from different patients. This study was the first report to compare the characterization of PDLPs and PDLSCs from the same person. The characterization of PDLPs and PDLSCs includes flow cytometry analysis, cell proliferation assay and the assessment of the colony-forming unit fibroblast. The osteogenic differentiation was evaluated by alkaline phosphatase activity, biomineralization (alizarin red staining) and gene expression of osteogenic markers. The adipogenic differentiation was examined by Oil Red O staining and adipocyte-related gene expression. Mesenchymal stem cell marker expression and colony-forming unit fibroblast analysis of PDLPs and PDLSCs were similar. However, PDLSCs grew faster than PDLPs on days 3 and 5 of the cell proliferation assay. Both PDLPs and PDLSCs could differentiate into osteoblast and adipocyte-like cells. However, the mineralization of PDLSCs was stronger than that of PDLPs. The characteristics of undifferentiated PDL cells in our study were not significantly impacted by the isolation method. We assumed that both PDLPs and PDLSCs are valuable cell sources for periodontal regeneration. However, PDLSCs have a possible advantage for the regeneration of alveolar bone.

Novel keratin preparation supports growth and differentiation of odontoblast-like cells.

To fabricate a keratin hydrogel, characterize its functionality as a biomaterial and investigate the effects of keratin on growth and differentiation of odontoblast-like cells. Keratins were extracted from sheep wool using a well-established technique. The extracted proteins were purified by dialysis, quantified by gel electrophoresis, mass spectrometry, amino acid analysis and inductively coupled mass spectrometry. The microstructure of the fabricated keratin hydrogels was studied by scanning electron microscopy, flow characteristics by rheometer, hydrolytic stability and cytocompatibility by Live/Dead(®) cell assay. Furthermore, the influence of keratin on odontoblast-like cells (MDPC-23) was assessed to confirm their bioactivity at different dilutions. Cell proliferation was studied using alamarBlue(®) assay and differentiation by alkaline phosphatase enzyme activity, alizarin red staining and calcium quantification, reverse transcription polymerase chain reaction (rt-PCR) and immunocytochemical staining for dentine matrix protein- 1 (DMP-1) expression. anova with Tukey's tests was performed for statistical comparison. The characterized hydrogel was injectable with a highly porous architecture that underwent slow degradation, and its cytocompatibility was statistically equivalent to collagen hydrogel (P > 0.05). Cell proliferation and differentiation were enhanced at the optimal keratin concentration of 0.1 mg mL(-1) . At this concentration, the influence of keratin on cell differentiation was demonstrated by marked elevation in alkaline phosphatase activity (P < 0.05), calcium deposition (P < 0.01), gene expression (P < 0.01) and positive immunostaining for DMP-1. The presence of keratin enhanced odontoblast cell behaviour. Keratin hydrogels may be a potential scaffold for pulp-dentine regen-eration.

Bone Tissue Engineering by Using Calcium Phosphate Glass Scaffolds and the Avidin–Biotin Binding System

Highly porous and interconnected scaffolds were fabricated using calcium phosphate glass (CPG) for bone tissue engineering. An avidin-biotin binding system was used to improve osteoblast-like cell adhesion to the scaffold. The scaffolds had open macro- and micro-scale pores, and continuous struts without cracks or defects. Scaffolds prepared using a mixture (amorphous and crystalline CPG) were stronger than amorphous group and crystalline group. Cell adhesion assays showed that more cells adhered, with increasing cell seeding efficiency to the avidin-adsorbed scaffolds, and that cell attachment to the highly porous scaffolds significantly differed between avidin-adsorbed scaffolds and other scaffolds. Proliferation was also significantly higher for avidin-adsorbed scaffolds. Osteoblastic differentiation of MG-63 cells was observed at 3days, and MG-63 cells in direct contact with avidin-adsorbed scaffolds were positive for type I collagen, osteopontin, and alkaline phosphatase gene expression. Osteocalcin expression was observed in the avidin-adsorbed scaffolds at 7days, indicating that cell differentiation in avidin-adsorbed scaffolds occurred faster than the other scaffolds. Thus, these CPG scaffolds have excellent biological properties suitable for use in bone tissue engineering.

MEK5 suppresses osteoblastic differentiation

Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells.We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts.Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis.

Temporal-controlled Dexamethasone Releasing Chitosan Nanoparticle System Enhances Odontogenic Differentiation of Stem Cells from Apical Papilla

IntroductionThe spatial and temporal control of stem cell differentiation into odontoblast-like cells remains one of the major challenges in regenerative endodontic procedures. The current study aims to synthesize and compare the effect of dexamethasone (Dex) release from 2 variants of Dex-loaded chitosan nanoparticles (CSnp) on the odontogenic differentiation of stem cells from apical papilla (SCAP). MethodsTwo variants of Dex-loaded CSnp were synthesized by encapsulation (Dex-CSnpI) and adsorption (Dex-CSnpII) methods. The physicochemical characterization of Dex-CSnpI and Dex-CSnpII was assessed by transmission electron microscopy, Zetasizer, and Fourier transform infrared spectroscopy, whereas the Dex release kinetics was assessed by spectrophotometry. A previously characterized SCAP cell line was cultured onto CSnp, Dex-CSnpI, or Dex-CSnpII. The biomineralization potential was determined by alizarin red staining. Alkaline phosphatase, dentin sialophosphoprotein, and dentin matrix protein-1 gene expressions were analyzed by real-time reverse-transcription polymerase chain reaction. ResultsDex-CSnpI resulted in slower release of Dex compared with Dex-CSnpII, but both demonstrated sustained release of Dex for 4 weeks. Biomineralization of SCAP was significantly higher (P < .05) in presence of Dex-CSnpII compared with that in Dex-CSnpI at 3 weeks. Alkaline phosphatase gene expression was significantly higher in the presence of Dex-CSnpII compared with Dex-CSnpI, with peak expression seen at 2 weeks (P < .05). The expression of odontogenic specific marker dentin matrix protein-1 was significantly higher in presence of Dex-CSnpII compared with Dex-CSnpI at 3 weeks (P < .05). ConclusionsCollectively, these data suggest that sustained release of Dex results in enhanced odontogenic differentiation of SCAP. These findings highlight the potential of temporal-controlled delivery of bioactive molecules to direct the spatial- and temporal-controlled odontogenic differentiation of dental stem cells.

Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture.

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7-21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells.

Evaluation of Corrosion Mechanisms at the Bone-Metal Interface of Hip Implants

Total hip replacements (THR) are considered to be a successful surgical intervention in orthopedic community. In a hip joint environment, the orthopedic metal implants are exposed to a severe degradation process that takes place at the implant-bone interface. The harsh chemical-biological milieu caused by the biological fluids surrounding the contact area may result in severe corrosion damages of the metallic alloys. Such effects were found to be one of the sources of early fracture, inflammation, metal ion release to the host, further loosening of the hip implant, thus limiting the durability of the implant. In this way, the proliferation and differentiation of osteoblastic precursor cells in Ti6Al4V alloys may be compromised, interfering with the continuous osseointegration processes of bone formation around the implant. The aim of this work is to investigate the corrosion behavior of Ti6Al4V alloys with the osseointegration process of osteoblastic cells (bone-forming cells) cultured on Ti-based materials, commonly used in uncemented femoral stems. The influence of these organic materials on the performance of these alloys is evaluated using an electrochemical approach. Ti6Al4V discs were mechanically polished using a series of SiC grinding papers up to a mirror finish (Ra < 40 nm). Then samples were sterilized in 70% alcohol during 1h. The non-colonized samples were tested with the sterilized polished surface. Other samples were then cultured with MG-63 osteoblastic-like cells in a culture medium: alpha-MEM supplemented with 10% fetal bovine serum and 1% penicillin-strepsin, pH of 7.4, at 37ºC in a humidified atmosphere of 5% CO2/air. Cell cultures on the colonized materials and control groups (polystyrene culture dish) were characterized through cell viability/proliferation, alkaline phosphatase activity, gene expression and respective morphology after 6 days of culture. All samples were analyzed by scanning electron microscopy (SEM), fluorescence microscopy and white light interferometry before and after the corrosion tests. Corrosion tests were performed using a three-electrode electrochemical cell configuration with a saturated calomel electrode (SCE) as the reference electrode (RE), a graphite rod as the counter electrode (CE), and the metallic alloy as the working electrode (WE). Three electrolytes were used for comparison: a common saline solution, phosphate buffer solution (PBS); and the culture media (CM) used for the cell cultures. All tests were performed inside an incubator in a controlled environment of temperature (37⁰C) and CO2 (5%). The open circuit potential was monitored during 1 hour, followed by an electrochemical impedance spectroscopy (EIS), in a frequency range from 10 KHz to 0.01 Hz with a ± 10 mV synovial amplitude, at a rate of 10 frequency per decade. The potentiodynamic scan was performed from -0.5 V to 1.5 V. The data was acquired with a Gamry Interface 1000 potentiostat. The results showed that when the surface of the Ti6Al4V alloy was exposed to an electrolyte containing organic material (culture media), the corrosion potential (Ecorr) decreased to -0.32 V, when compared to the surface exposed to a saline solution rich in phosphates (PBS), -0.22 V. Consequently, the passive current density (Ipass) also decreased, from 3.06 X 10-6 A/cm2 in PBS to 1.95X 10-6 A/cm2 in culture media. On the other hand, when the Ti6Al4V was colonized with osteoblastic cells, maintaining the electrolyte rich in organic material (culture media), apparently, this values were always higher than former conditions. The colonized surfaces have shown a higher corrosion potential of the metallic surface with a quicker formation of the passive film with higher polarization resistance. The simultaneous presence of osteoblastic cells and serum proteins in the media might induce a higher passivation rate of the oxide film, promoting a higher resistance to corrosion of the surface of the material. These findings bring a better understanding on the correlation between the corrosion mechanisms happening at the surface of the metallic implants and the cellular responses to its products.

In Situ Forming Hydrogel Based on Chondroitin Sulfate–Hydroxyapatite for Bone Tissue Engineering

In this research, tissue engineered hydrogels consist of chondroitin sulfate and hydroxyapatite capable of light curing and cell encapsulation were prepared. Chemical and physical characteristics were analyzed by FTIR spectroscopy, compressive strength and water uptake measurements. Observations with fluorescence microscopy as well as MTT viability assay revealed no toxicity for encapsulated cells. Moreover, alkaline phosphatase activity, calcium deposition, and gene expression analysis were determined to prove the osteogenic potential of the prepared hydrogel. In conclusion, this in situ forming hydrogel can be utilized as filler for bone deficiencies as well as carrier for cell encapsulation and osteogenic differentiation.

MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth.

Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner.

Icariin, a flavonoid isolated from Epimedium pubescens, has previously been identified to exert beneficial effects on preventing bone loss and promoting bone regeneration. However, molecular mechanisms for its anabolic action have, up to now, remained largely unknown. Effects of icariin on cell proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) were systematically evaluated. To characterize underlying mechanisms, its effects on mitogen-activated protein kinase (MAPK) signalling pathways were determined. Results showed that icariin might not have enhanced effects on cell proliferation. However, it seemed to significantly enhance osteogenic differentiation of BMSCs, demonstrated by increasing alkaline phosphatase (ALP) activity and gene expression of collagen type I (Col I), osteocalcin (OCN) and osteopotin (OPN). It was demonstrated that icariin rapidly phosphorylated extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N terminal kinase (JNK). Furthermore, icariin-stimulated osteogenic effects on BMSCs were dramatically attenuated by treatment with either specific ERK inhibitor of PD98059, p38 inhibitor of SB202190 or JNK inhibitor SP600125. These results provide a potential mechanism of anabolic activity of icariin on BMSCs involving ERK, p38 and JNK MAPK pathways.

Osteogenic Potential of Icariin Compared with Recombinant Human Bone Morphogenetic Protein 2 In Vitro: A Preliminary Study

Icariin, the primary active ingredient of Herba Epimedii which has been used for decades to treat bone related maladies in China, has the ability to support bone regeneration. In this study, we investigated icariin's potential to stimulate osteogenesis using an in vitro studies to compare icariin's ability as a function of time and dose to induce osteoblastogenesis in MC3T3-E1 cells with that of recombinant human bone morphogenetic protein 2 (rhBMP2). The optimal concentration of icariin in stimulating the proliferation of MC3T3-E1 was 10-5 M. There is no significant difference between icariin and rhBMP2 relative to cell proliferation, alkaline phosphatase (ALP) activity, and osteocalcin (OCN) expression. Icariin had better ALP activity after a 7 day culture and OCN expression after 4 and 10 day cultures compared with rhBMP2. Moreover, icariin produced more mineralized nodules of a larger area than rhBMP2. However, compared with rhBMP2, icariin only showed a significantly higher ALP gene expression on day 4, a 3 fold BMP2 expression on day 7, and Col 1 expression on day 4. In conclusion, compared with rhBMP2, the results indicate that icariin could be a good option for bone regeneration clinically, since it is an inexpensive and safe drug with an excellent osteogenesis potential.

Icariin induced proliferation and differentiation of MC3T3-E1 osteoblasts via ERK and JNK signaling coupled with estrogen receptor

Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation. (Chin J Endocrinol Metab, 2015, 31: 148-154) Key words: Icariin; Osteoblasts; Estrogen receptor; Mitogen-activated protein kinase

Pulsed laser deposition of magnesium-doped calcium phosphate coatings on porous polycaprolactone scaffolds produced by rapid prototyping

Polycaprolactone (PCL) is a biodegradable and biocompatible polyester whose low melting point facilitates production of 3D porous scaffolds with precisely defined dimensions and internal architecture by rapid prototyping techniques. To improve the suitability of such PCL scaffolds for bone regeneration applications, they were coated with inorganic layers of calcium phosphate (CaP) and CaP doped with 0.6% w/v magnesium (CaP+Mg) using pulsed laser deposition (PLD) and characterized in vitro using osteoblast-like Saos-2 cells.Saos-2 cells were able to adhere to all scaffolds. CaP+Mg coatings significantly increased activity of alkaline phosphatase (ALP), an early differentiation marker, after 7 days. However, gene expression of ALP after 7 days was markedly lower on the same scaffolds.These data show the feasibility of coating PCL with CaP layers by PLD and the possibility of influencing osteoblastic differentiation by magnesium doping of the CaP coating.

Nanotubes Functionalized with BMP2 Knuckle Peptide Improve the Osseointegration of Titanium Implants in Rabbits.

To determine the effects of surface modification on implant osseointegration in vivo, we first immobilized polydopamine onto 70-nm diameter TiO2 nanotubes as an intermediate layer, and then conjugated a 21 amino acid peptide sequence (the so-called "knuckle peptide") of bone morphogenetic protein-2 (BMP-2) onto the nanotubes created by electrochemical anodization. We inserted these implants into the tibiae of rabbits and measured the gene expression and bone formation around them. The successful fabrication of BMP-2 knuckle peptide was confirmed by contact angle measurement, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The histological features and the amount of bone fluorescence around the implants on non-decalcified sections were investigated at 3, 5, 8, and 12 weeks after implantation using traditional light and fluorescence microscopy, and the gene expression of alkaline phosphatase, osterix, collagen-I, and tartrate-resistant acid phosphatase was examined by real-time PCR at 1, 2, 3, 4, and 5 weeks after implantation. The results demonstrated a significant increase in bone-implant contact, quantity of fluorescence, and gene expression levels of the bone attached to implants with immobilized BMP-2 knuckle peptide compared with the other two control groups. In conclusion, the surface functionalization of TiO2 nanotubes with BMP-2 knuckle peptide was beneficial for osseointegration and this approach could be further developed to improve Ti-based implants for various applications.

Dietary saponins and phytosterols do not affect growth, intestinal morphology and immune response of on-growing European sea bass (Dicentrarchus labrax )

This study was the first to investigate the effect of soy antinutrients, saponins and phytosterols, in on-growing sea bass. Seven diets were formulated: a control diet (fishmeal and fish oil based) and six diets containing 1 or 2 g kg−1 levels of soya saponins, 5 or 10 g kg−1 levels of phytosterols or a combination of 1 g kg−1 saponins + 5 g kg−1 phytosterols or 2 g kg−1 saponins + 10 g kg−1 phytosterols. After a 59 days feeding trial fish were weighed, faeces collected for digestibility studies and plasma sampled for cholesterol quantification. At day 15 and 59, distal intestine (DI) samples were collected for histological evaluation and quantification of maltase (Malt), alkaline phosphatase (AlP) and leucine aminopeptidase (LAP) activities. At day 59, DI was collected for gene expression of AlP, Malt and immune related genes. Antinutrients had no effect on fish performance, digestibility, plasma cholesterol levels and digestive enzymes activities and gene expression. Only mild morphological changes were present in groups fed high levels of saponins + phytosterols. Expression of immune related genes showed no differences between groups. Overall, on-growing sea bass showed high tolerance to dietary antinutrients up to the levels tested.

St. John's Wort (Hypericum perforatum) stimulates human osteoblastic MG-63 cell proliferation and attenuates trabecular bone loss induced by ovariectomy.

BACKGROUND/OBJECFTIVESThe effect of St. John's Wort extract (SJW) on MG-63 cell proliferation and trabecular bone loss induced by ovariectomy was examined.MATERIALS/METHODSProliferation, expression of estrogen receptor (ER) α and ER β, and gene expressions of osteoprotegerin (OPG), osteocalcin (OC) and alkaline phosphatase (ALP) were examined in MG-63 cells treated with or without SJW. Ovariectomized rats were treated with SJW at the dose of 100 or 200 mg/kg/day, β-estradiol-3-benzoate (E2), or vehicle only (OVX-C), and sham operated rats were treated with vehicle only (Sham-C). Serum ALP and C-telopeptide (CTX), and femoral trabecular bone loss were examined.RESULTSSJW increased MG-63 cell proliferation and expression of ER α and ER β, and positive effect was shown on gene expressions of ALP, OC and OPG. SJW also showed estrogen like effect on bone associated with slowing down in trabecular bone loss. Histopathology by H&E showed rats treated with SJW displayed denser structure in metaphyseal region of distal femur compared with rats in OVX-C. SJW was shown to reduce serum CTX in OVX rats.CONCLUSIONThe present study provides new insight in preventing estrogen deficiency induced bone loss of SJW and possibility for its application in bone health supplement.

FOXO1 Is Involved in the Effects of Cigarette Smoke Extract on Osteoblastic Differentiation of Cultured Human Periosteum-derived Cells.

Cigarette smoke is associated with delayed fracture healing, alterations in mineral content, and osteoporosis, however, its effects on osteoblastic differentiation of osteoprogenitor cells are not fully understood. In the present study, we examined the effects of cigarette smoke extract (CSE) on osteoblastic differentiation of cultured human periosteum-derived cells. We found that CSE inhibited alkaline phosphatase (ALP) activity, mineralization and Runx2 transactivation of the periosteum-derived cells. Nucleofection of RUNX2 into the periosteum-derived cells increased expression of endogenous osteocalcin (OC) and ALP genes in osteogenic induction medium and increased OC expression in non-osteogenic medium. Treatment of the periosteum-derived cells with CSE resulted in decreased phosphorylation of AKT and forkhead box protein O1 (FOXO1). The AKT phosphorylation-resistant mutant, FOXO1-A3, inhibited transcriptional activity of RUNX2 in the periosteum-derived cells. The current study suggests one mechanism by which CSE exposure leads to inhibition of osteoblastic differentiation of cultured human periosteum-derived cells.

Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration.

Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs.