Upregulation Of Alkaline Phosphatase Research Articles

Synergistic effects of dual growth factor delivery from composite hydrogels incorporating 2-N,6-O-sulphated chitosan on bone regeneration

A promising strategy to accelerate bone generation is to deliver a combination of certain growth factors to the integration site via a controlled spatial and temporal delivery mode. Here, a composite hydrogel incorporating poly(lactide-co-glycolide) (PLGA) microspheres was accordingly prepared to load and deliver the osteogenic rhBMP-2 and angiogenic rhVEGF165 in the required manner. In addition, 2-N,6-O-sulphated chitosan (26SCS), which is a synergetic factor of growth factors, was incorporated in the composite hydrogel as well. The system showed a similar release behaviour of the two growth factors regardless of 26SCS inclusion. RhBMP-2 loaded in PLGA microspheres showed a sustained release over a period of 2 weeks, whereas rhVEGF165 loaded in hydrogel eluted almost completely from the hydrogel over the first 16 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. Moreover, an enhanced cell response was achieved upon the delivery of dual growth factors, compared to that obtained with a single factor. Furthermore, in the presence of 26SCS, the system revealed significantly upregulated alkaline phosphatase activity, human umbilical vein endothelial cell proliferation, sprouting, nitric oxide secretion, and angiogenic gene expression. This study highlighted that the composite hydrogel incorporated with 26SCS appears to constitute a promising approach to deliver multiple growth factors. From our findings, we could also conclude that rhBMP-2 can promote angiogenesis and that the mechanism is worthy of further study in subsequent research.

Effect of surface charge on osteoblastic proliferation and differentiation on a poly(ethylene glycol)-diacrylate hydrogel

Two charge monomers, namely 2-(methacryloyloxy)ethyl-trimethylammonium chloride (MAETAC) and sodium methacrylate (SMA), were incorporated into poly(ethylene glycol)-diacrylate (PEGDA) hydrogels to investigate the effects of surface charge on the proliferation and differentiation of osteoblasts. The physicochemical properties of the polymers were characterized, and MC3T3-E1 cells were seeded on the hydrogels to evaluate the effect of charge polarity and density on osteoblastic proliferation and differentiation. FTIR results revealed that the two charged monomers were successfully incorporated into PEGDA. The zeta potential of the hydrogels became more positive or negative with increasing concentration of MAETAC or SMA. The zeta potential of the charged hydrogels remained constant after immersion in culture medium for different time points. Other physicochemical properties such as surface morphology, swelling ratio in PBS, contact angle, and elastic modulus were not significantly different among each group with different concentrations of charge monomers incorporated into PEGDA. The modification of hydrogels with charge monomers not only improved osteoblastic proliferation but also upregulated alkaline phosphatase activity and the expression of osteogenic marker genes and relative growth factors. These findings indicate that, in contrast to charge polarity, the charge density would be more important to improve osteoblast-like cells proliferation and differentiation on the poly(ethylene glycol)-diacrylate hydrogel. The hydrogel can be designed by controlling the incorporation of charge monomers. This study provides a model to study the effect of charge on cell behavior.

Portland cement induces human periodontal ligament cells to differentiate by upregulating miR-146a.

Bioaggregates such as Portland cement (PC) can be an economical alternative for mineral trioxide aggregate (MTA) with additional benefit of less discoloration. MTA has been known to induce differentiations of several dental cells. MicroRNAs are important regulators of biological processes, including differentiation, physiologic homeostasis, and disease progression. This study is to explore how PC enhances the differentiation of periodontal ligament (PDL) cells in microRNAs level. PDL cells were cultured in a regular PC- or MTA-conditioned medium or an osteoinduction medium (OIM). Alizarin red staining was used to evaluate the extent of mineralization. Transfection of microRNA mimics induced exogenous miR-31 and miR-146a expression. The expression of microRNAs and differentiation markers was assayed using reverse-transcriptase polymerase chain reaction. PC enhanced the mineralization of PDL cells in a dose-dependent manner in the OIM. Exogenous miR-31 and miR-146a expression upregulated alkaline phosphatase (ALP), bone morphogenic protein (BMP), and dentin matrix protein 1 (DMP1) expression. However, miR-31 and miR-146a modulates cementum protein 1 (CEMP1) expression in different ways. PC also enhanced ALP and BMP but attenuated CEMP1 in the OIM. Although the OIM or PC treatment upregulated miR-21, miR-29b, and miR-146a, only miR-146a was able to be induced by PC in combination with OIM. This study demonstrated that PC enhances the differentiation of PDL cells, especially osteogenic through miR-146a upregulation. In order to control the ankylosis after regenerative endodontics with the usage of bioaggregates, further investigations to explore these differentiation mechanisms in the miRNA level may be needed.

Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells

Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 μM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 μM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 μM) significantly elevated TEER at 6–24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 μM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1.

Modulation of osteoblast behavior on nanopatterned yttria-stabilized zirconia surfaces

This study utilizes the technique of self-assembly to fabricate arrays of nanoislands on (001)-oriented yttria-stabilized zirconia single crystal substrates with miscut of 10° toward <110> direction. These self-assembled nanostructures were annealed at 1100°C for 5h upon doping with 10mol% gadolinium-doped ceria (GDC) by powder-suspension based method. X-Ray diffraction result showed that the miscut substrate after doping GDC was in the cubic phase. Energy dispersive X-ray (EDX) illustrates that the nanopatterned material contains all the elements from the GDC source and yttria-stabilized zirconia (YSZ) substrate. It also demonstrates a higher surface roughness and a more hydrophilic surface. The nanostructured materials were subsequently used for an in vitro study using a human fetal osteoblastic cell line (hFOB). An improved spreading, enhanced cell proliferation and up-regulated alkaline phosphatase activity (ALP) were observed on the nanopatterned substrates compared to the control substrates. Calcium deposits, which were stained positively by Alizarin Red S, appeared to be more abundant on the nanopatterned surfaces on day 7. The overall findings suggest that post fabrication treatment with surface modification such as creating a nanostructure (e.g. nanopatterns) can improve biocompatibility.

Porcine placenta hydrolysates enhance osteoblast differentiation through their antioxidant activity and effects on ER stress

BackgroundOsteoporosis is a disease characterized by decreased bone strength, decreased bone mass, and bone deterioration. Oxidative damage is an important contributor to functional changes in the development of osteoporosis. Here we found that porcine placenta hydrolysates (PPHs) protect MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative damage.MethodsIn vitro cell viability was determined using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a commercially available caspase kit. ALP, osteocalcin, Runx2, and osterix expression levels were evaluated by RT-PCR using isolated RNA. ROS, NADPH oxidase, and SOD activity levels were also measured.ResultsWe investigated the mechanisms underlying PPH-mediated inhibition of H2O2-induced ER stress and ROS production. PPHs also regulated osteoblast differentiation via the upregulation of alkaline phosphatase (ALP) expression in MC3T3-E1 osteoblastic cells. Also, treatment with PPHs enhanced the transcription of osteocalcin, Runx2, and osterix. These effects were all associated with the antioxidant actions of PPHs. Moreover, PPHs reversed the decrease in SOD activity, decreased ROS release, and inhibited NADPH oxidase activity in H2O2-treated MC3T3-E1 osteoblastic cells.ConclusionsPPHs protect cells against H2O2-induced cell damage when ER stress is involved. In addition, PPHs enhance osteoblast differentiation. This enhancement likely explains the regulatory effect of PPHs on bone metabolism disturbances, i.e. PPHs control ER stress and the related ROS production in osteoblasts.

Longan (Dimocarpus longan Lour.) Fruit Extract Stimulates Osteoblast Differentiation via Erk1/2-Dependent RUNX2 Activation.

Longan (Dimocarpus longan Lour.) has been used as a traditional oriental medicine and possesses a number of physiological activities. In this study, we used cell-based herbal extract screening to identify longan fruit extract (LFE) as an activator of osteoblast differentiation. LFE up-regulated alkaline phosphatase (ALP) activity, induced mineralization, and activated Runx2 gene expression in MC3T3-E1 cells. Furthermore, treatment of MC3T3-E1 cells with LFE promoted the phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2); however, abrogation of Erk1/2 activation with PD98059 resulted in down-regulation of the phospho- SMAD1/5/8 and Runx2 levels, which in turn reduced the ALP activity. Our findings suggest that LFE exerts its osteogenic activity through activation of the ERK signaling pathway and may have potential as an herbal therapeutic or a preventive agent for the treatment of osteoporosis.

Characterization of age- and gender-specific trabecular bone structural parameters and mesenchymal stromal cells of osteoarthritic facet joints in lumbar spinal stenosis

Purpose: Facet joint osteoarthritis may be a cause of low back pain in degenerative spine diseases such as lumbar spinal stenosis. While increased bone remodeling is a clinical hallmark of osteoarthritis, the trabecular bone structural parameters of osteoarthritic lumbar facet joints have not been characterized thus far. Interestingly, an age- and gender-specific decrease of trabecular number, thickness and volume fraction has been previously described for lumbar 4/5 facet joints of healthy individuals. Here, we sought to characterize trabecular bone structural parameters and subchondral bone-resident mesenchymal stromal cells (MSC) of osteoarthritic facet joints and compare these with regard to age, gender and healthy controls. Methods: Twenty-one patients scheduled for decompression and stabilization surgery for degenerative spinal stenosis were recruited for this study. Facet joint specimens were harvested during surgery and either fixed in formalin for microcomputed tomography (n = 15) or isolation of MSC by collagenase digestion (n = 6). MicroCT was performed spatial resolution of 20.5 μm. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were evaluated using CT Analyser. Trabecular bone structural parameters of healthy subjects were taken from Wilke and co-workers (J. Anat. 2012). Statistical analysis of differences between gender and age groups (40–60 and 60–80 years) were performed using two-way analysis of variance (ANOVA). Early passage MSC were subjected to adipogenic and osteogenic differentiation for three weeks and evaluated using alkaline phosphatase assay, Alizarin red and Oil red O staining and RT-PCR. Results: Trabecular BV/TV was 0.36 ± 0.14 and 0.35 ± 0.17 for osteoarthritic lumbar facet joints of male and female individuals, respectively. Contrastingly, mean BV/TV in age-matched healthy controls was 0.32 (male) and 0.25 (female). Tb.Th was in the same range for both genders and age-groups (overall mean 0.20 ± 0.07 mm) and did not differ from healthy controls. The overall Tb.Sp averaged 0.52 ± 0.16 mm, which was lower than averages observed for age-matched healthy females (0.60) and males (0.71). Accordingly, Tb.N in osteoarthritic facet joints of male (1.9 ± 1.3/mm) and female (1.7 ± 0.8/mm) patients with lumbar spinal stenosis was higher than reported for healthy controls (1.5–1.0/mm). Statistical analysis did not reveal any age- or gender-specific differences in trabecular bone structural parameters of osteoarthritic lumbar facet joints. Facet joint MSC showed a strongly upregulated alkaline phosphatase activity upon adipogenic (8.1-fold) and osteogenic (4.6-fold) differentiation. Only a low number of lipid vacuoles were detected after adipogenesis. Alizarin red staining revealed varying degrees of in vitro matrix mineralization in MSC, which ranged from blunted to strong mineralization. Conclusions: Subchondral trabecular bone remodeling in lumbar facet joint osteoarthritis is characterized by an increase in bone volume fraction due to a higher trabecular number, but not an increase of trabecular thickness. MSC from osteoarthritic facet joints display impaired adipogenesis and abnormal mineralization during osteogenesis. These data provide a strong rationale for targeting increased bone remodeling in lumbar facet joint osteoarthritis.

Development of Biodegradable Poly(citrate)-Polyhedral Oligomeric Silsesquioxanes Hybrid Elastomers with High Mechanical Properties and Osteogenic Differentiation Activity

Biodegradable elastomeric biomaterials have attracted much attention in tissue engineering due to their biomimetic viscoelastic behavior and biocompatibility. However, the low mechanical stability at hydrated state, fast biodegradation in vivo, and poor osteogenic activity greatly limited bioelastomers applications in bone tissue regeneration. Herein, we develop a series of poly(octanediol citrate)-polyhedral oligomeric silsesquioxanes (POC-POSS) hybrids with highly tunable elastomeric behavior (hydrated state) and biodegradation and osteoblasts biocompatibility through a facile one-pot thermal polymerization strategy. POC-POSS hybrids show significantly improved stiffness and ductility in either dry or hydrated conditions, as well as good antibiodegradation ability (20-50% weight loss in 3 months). POC-POSS hybrids exhibit significantly enhanced osteogenic differentiation through upregulating alkaline phosphatase (ALP) activity, calcium deposition, and expression of osteogenic markers (ALPL, BGLAP, and Runx2). The high mechanical stability at hydrated state and enhanced osteogenic activity make POC-POSS hybrid elastomers promising as scaffolds and nanoscale vehicles for bone tissue regeneration and drug delivery. This study may also provide a new strategy (controlling the stiffness under hydrated condition) to design advanced hybrid biomaterials with high mechanical properties under physiological condition for tissue regeneration applications.

Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth

ObjectiveThe aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. DesignNotch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. ResultsThe isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. ConclusionThe surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.

Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

AIM:Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).METHODS:Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.RESULTS:In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.CONCLUSION:Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

N16, a Nacreous Protein, Inhibits Osteoclast Differentiation and Enhances Osteogenesis.

N16 is a protein from the nacreous layer of Pinctada fucata, a pearl oyster. It has been found to promote biomineralization, and we hypothesized that it also plays a role in bone metabolism. The cDNA of N16 was cloned and expressed in Escherichia coli to produce N16 protein, which was purified to high homogeneity by ion-exchange and gel filtration columns. The effects of N16 on osteoclast differentiation and osteogenesis were clarified using the murine preosteoclast cell line RAW 264.7 and the preosteoblast cell line MC3T3-E1. Results on preosteoclasts showed that N16 only slightly inhibited cell survival but significantly inhibited differentiation induced by receptor activator of nuclear factor kappa-B ligand (RANKL). Apart from reduced formation of multinucleated osteoclasts, N16-treated cells exhibited lower gene expression and enzymatic activity typical of mature osteoclasts. Actin ring formation and intracellular acidification essential for osteoclastic function were also impaired upon N16 treatment. At concentrations nontoxic to preosteoblasts, N16 strongly up-regulated alkaline phosphatase activity and increased mineralized nodule formation, which are indicative of differentiation into osteoblasts. These effects coincided with an increase in mRNA expression of osteoblast markers osteopotin and osteocalcin. The present study demonstrated that N16 has both anabolic and antiresorptive effects on bone, which makes it potentially useful for treating osteoporosis.

Functionalization of silk fibroin through anionic fibroin derived polypeptides

Event Abstract Back to Event Functionalization of silk fibroin through anionic fibroin derived polypeptides Gabriele Griffanti1, 2, Mark James-Bhasin1, Ilaria Donelli3, Giuliano Freddi3 and Showan Nazhat1 1 McGill University, Department of Mining and Materials Engineering, Canada 2 Universita degli Studi di Milano, Dipartimento di Biotecnologie e Bioscienze, Italy 3 Stazione Sperimentale per la Seta, Italy Introduction: Silk fibroin (SF) is frequently considered as a template for mimicking biomineralization[1]. The α-chymotrypsin digestion of aqueous SF solutions generates anionic fibroin derived polypeptides (Cs)[2], which rapidly induce carbonated-hydroxyapatite (CHA) formation in dense collagen gels[3],[4]. Herein, it is hypothesized that the incorporation of Cs into SF would induce CHA deposition. The effect of Cs incorporation on the mineralization of electrospun (ES) SF scaffolds was assessed in simulated body fluid (SBF), and its potential role in mediating the osteoblastic differentiation of seeded mesenchymal stem cells (MSCs) was investigated. Materials and Methods: Cs [2] incorporated ES-SF scaffolds (at 1, 2, 5 and 25 wt.% Cs) were produced[5]. Zeta potential and contact angle measurements were used to investigate the effect of Cs on scaffold surface charge and hydrophilicity. Mineralization of the various scaffolds was assessed at days 1, 3 and 7 in 1.5 X Kokubo’s SBF[6]. Mineralization extent was assessed using ATR-FTIR, XRD and SEM. Passage 9 murine bone marrow derived MSCs were seeded at a density of 5x103 cells/cm2 on ES-SF scaffolds and cultured for 21 days in basal and osteogenic media. Alamar BlueTM assay was carried out to investigate seeded MSC proliferation at days 1, 7, 14 and 21 in culture. Quantitative real-time PCR was carried out at days 14 and 21 to assess MSC osteoblastic differentiation. Results: There was an increase in the negative charge properties of the ES-SF scaffolds with increasing Cs content (FIG 1, A). Immersion in SBF indicated CHA formation, increasing with Cs content (Fig. 1, B-C). Seeded MSCs attached on all scaffold types (Fig. 2) with differences in metabolic activities when cultured in osteogenic medium. MSCs on Cs incorporated ES-SF scaffolds demonstrated higher metabolic activities compared to those seeded on neat ES-SF (Fig. 3, A-B). Relative to basal medium, there was an up-regulation of alkaline phosphatase, Runx2 and Osteocalcin in osteogenic medium (at days 14 and 21). Surprisingly, under basal medium, MSCs seeded on Cs incorporated ES-SF scaffolds also expressed both Runx2 and Osteocalcin (FIG. 3, C-E). Discussion: In order for the onset of mineralization to occur, complexes such as –COOCa+ and (–COO)2Ca are required to form on the surface of SF. It is speculated that the carboxyl groups provided by the incorporation of negatively charged Cs polypeptides into SF served as nucleation sites for CHA deposition. Furthermore, Cs incorporation could be considered as a novel route to modulate MSC osteoblastic differentiation in the absence of osteogenic conditioning. Conclusions: The ability to incorporate Cs during SF fibre formation can induce CHA formation and control MSC osteoblastic differentiation, providing a basis for future optimization of ES-SF scaffolds for potential bone tissue engineering applications. Funding of CIHR, NSERC, CFI, Quebec MEIE, McGill University Faculty of Engineering Gerald Hatch Faculty Fellowship, MEDA and Università degli studi di Milano - Bicocca in collaboration with Assolombarda are gratefully acknowledged.

BMP-2 Induced Expression of PLCβ1 That is a Positive Regulator of Osteoblast Differentiation.

Bone morphogenetic protein 2 (BMP-2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide-phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP-2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up-regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic-PLCβ1mutant (cyt-PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR-214 is a regulator of Osterix (Osx) which is an osteoblast-specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR-214 in the control of osteogenic differentiation by gain- and loss- of function experiment. The results indicated that inhibition of miR-214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP-2-induced osteogenesis by targeting PLCβ1.

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation.

BackgroundTridax procumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts.ResultsTPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2.ConclusionOur findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Stimulation with bone morphogenetic protein-2 (BMP-2) enhances bone–tendon integration in vitro

ABSTRACTPurpose: Preclinical studies have reported that bone morphogenetic protein (BMP)-2 promotes bone–tendon healing following anterior cruciate ligament reconstruction. We examined the region-specific effects of BMP-2 on osteoblast and fibroblast differentiation in a highly standardized murine in vitro co-culture model of bone–tendon integration. Materials and methods: We used quantitative PCR to measure the dose- and time-dependent influence of BMP-2 on the expression of alkaline phosphatase, osteocalcin, collagen type 1 (alpha 1 chain), runt-related transcription factor 2, osteopontin, collagen type 1 (alpha 2 chain), collagen type 5 (alpha 1 chain), decorin, fibromodulin, mohawk homeobox, bone morphogenetic protein receptor, type 1A, bone morphogenetic protein receptor, type 2, and Noggin in the osteoblast, interface, and fibroblast regions of a co-culture model of the murine preosteoblast cell line MC3T3-E1 and the fibroblast cell line 3T6. Results: Stimulation with BMP-2 resulted in a significant upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), collagens (p < 0.001), runt-related transcription factor 2 (p < 0.05), and osteopontin (p < 0.001) expression in the osteoblast region. In the interface region, BMP-2 exposure led to dose- and time-dependent upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), osteopontin (p < 0.001), runt-related transcription factor 2 (p < 0.001), and markers of extracellular matrix production (p < 0.001). Both BMP receptors showed a significant BMP-2-dependent upregulation at the interface region, and Noggin was downregulated at the osteoblast and interface region following BMP-2 exposure. Conclusions: Exposure to BMP-2 upregulated the expression of genes associated with bone–tendon integration in vitro, suggesting the stimulation of transdifferentiation processes at the interface and fibroblast regions as well as the induction of positive feedback mechanisms. Further studies will be needed to establish BMP-2 dose and treatment algorithms following tendon reinsertion and reconstruction.

Cyclic stretch enhances bone morphogenetic protein-2-induced osteoblastic differentiation through the inhibition of Hey1.

Substantial evidence has indicated that osteoblastic differentiation may be regulated by mechanical loads or bone morphogenetic protein-2 (BMP-2). BMP-2-induced in vivo osteogenesis can be significantly enhanced in the presence of mechanical stimuli, revealing the therapeutic potential of the combined application of BMP-2 and mechanical loads in clinical bone diseases (e.g., bone fractures and osteoporosis); however, the underlying mechanisms remain elusive. In this study, we found that cyclic stretch or BMP-2 alone increased the expression of osteoblastic differentiation markers, including alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), as shown by RT-qPCR, western blot analysis and ALP activity test. Furthermore, our results revealed that cyclic mechanical stretch with 10% elongation at 0.1 Hz significantly enhanced the BMP-2-induced upregulation of ALP and Runx2 expression in osteoblast-like MC3T3-E1 cells. Cyclic stretch also inhibited the BMP-2-induced upregulation of Hes-related family bHLH transcription factor with YRPW motif 1 (Hey1, measured by RT-qPCR and immunofluorescence staining), a potent negative regulator of osteogenesis. Moreover, the transient transfection of a Hey1 expression plasmid (pcDNA3.1-Hey1) significantly reversed the effects of cyclic stretch on the BMP-2-induced upregulation of differentiation markers in the MC3T3-E1 cells. This revealed the importance of Hey1 in modulating BMP-2-induced osteoblastic differentiation in response to cyclic stretch. Taken together, our results demonstrated that cyclic stretch enhanced the BMP-2-induced osteoblastic differentiation through the inhibition of Hey1. The present study broadens our fundamental knowledge of osteoblastic mechanotransduction and also sheds new insight into the mechanisms through which the combined application of BMP-2 and mechanical load promotes osteogenesis.

Protective Role of Smad6 in Inflammation-Induced Valvular Cell Calcification.

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-α) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle α actin, and negative for an endothelial cell marker. TNF-α upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-α significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-α-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-α further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-α induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6.

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.

Calcium silicate/calcium phosphate biphasic cements for vital pulp therapy: chemical-physical properties and human pulp cells response.

The aim was to test the properties of experimental calcium silicate/calcium phosphate biphasic cements with hydraulic properties designed for vital pulp therapy as direct pulp cap and pulpotomy. CaSi-αTCP and CaSi-DCDP were tested for ion-releasing ability, solubility, water sorption, porosity, ability to nucleate calcium phosphates, and odontoblastic differentiation—alkaline phosphatase (ALP) and osteocalcin (OCN) upregulation—of primary human dental pulp cells (HDPCs). The materials showed high Ca and OH release, high open pore volume and apparent porosity, and a pronounced ability to nucleate calcium phosphates on their surface. HDPCs treated with CaSi-αTCP showed a strong upregulation of ALP and OCN genes, namely a tenfold increase for OCN and a threefold increase for ALP compared to the control cells. Conversely, CaSi-DCDP induced a pronounced OCN gene upregulation but had no effect on ALP gene regulation. Both cements showed high biointeractivity (release of Ca and OH ions) correlated with their marked ability to nucleate calcium phosphates. CaSi-αTCP cement proved to be a potent inducer of ALP and OCN genes as characteristic markers of mineralization processes normally poorly expressed by HDPCs. Calcium silicate/calcium phosphate cements appear to be attractive new materials for vital pulp therapy as they may provide odontogenic/dentinogenic chemical signals for pulp regeneration and healing, and dentin formation in regenerative endodontics.