Alkaline Phosphatase Secretion Research Articles

Multifunctional ZnO/TiO2 nanoarray composite coating with antibacterial activity, cytocompatibility and piezoelectricity

Abstract Metal oxides with nanostructures such as zinc oxide (ZnO), titanium dioxide (TiO2) have been used in biomedical fields for their multifunctional properties. In this study, ZnO/TiO2 nanoarray (nZnO/TiO2) coatings were prepared via hydrothermal synthesis followed by low temperature liquid phase method. The particle size of the composites were no more than 100 nm in diameter, assembled into nanoarray on the Ti substrate. In vitro antibacterial experiments showed that the maximum bacteriostatic rate could reach 99% against Staphylococcus aureus and 90% against Escherichia coli, respectively. Moreover, the nZnO/TiO2 coatings were of cytocompatibility and biocompatibility, promoting the proliferation of MC3T3-E1 and the expression of alkaline phosphatase (ALP). The piezoelectric properties of nZnO/TiO2 coatings were preliminarily investigated. The smaller the size of the composite particle was, the better the antibacterial property, biocompatibility and piezoelectric properties were. Under the stimulation of the periodic loading, the growth of MC3T3-E1 was promoted, so the secretion of ALP was. The nZnO/TiO2 composite coating with antibacterial activity, osteogenesis and intellectual stimulation would be a promising smart coating for orthopedic implants.

Potential of an electrospun composite scaffold of poly (3-hydroxybutyrate)-chitosan/alumina nanowires in bone tissue engineering applications

The choice of material types for tissue engineering scaffolds and the design of methods are contributive in yielding the proper result. In this study, 1–5% wt. Alumina nanowires are added to (Polyhydroxybutyrate-Chitosan) PHB-CTS alloy solution, and the scaffolds are prepared by electrospinning method. The fiber diameters, porosity percentages and uniform distribution of Alumina nanowires are assessed by SEM, EDS and TEM. The surface roughness of the fibers is confirmed by FESEM and AFM. The crystallinity of nanofibers is calculated by DSC and verified by FTIR. The tensile strength of the PHB-CTS scaffold increase up to >10 fold in presence of 3% wt. Alumina. Formation of calcium phosphate sediments only on the surface of Alumina containing scaffolds after 7 and 28 days of immersion in SBF is observed by SEM, and verified by XRD analysis. Proliferation and viability of MG-63 cells and alkaline phosphatase secretion are significantly higher on scaffolds containing Alumina than that of the PHB or PHB-CTS. The appropriate properties of Alumina which affected in cell behavior, hydrophilicity enhancement, bioactivity and mechanical properties make it contribution agent in bone tissue engineering.

Scalable MSC-derived bone tissue modules: In vitro assessment of differentiation, matrix deposition, and compressive load bearing

Enhancements to the mechanical properties of modular designs for bone tissue engineering could increase their clinical applications. In this study, bone marrow mesenchymal stem cells (MSCs) and hydroxyapatite (HAP) microgranules were encapsulated in polyelectrolyte complex membranes composed of chondroitin 4-sulfate (C4S), carboxymethyl cellulose (CMC) and chitosan. Microcapsules were formed with and without HAP microgranules, and cultured in either osteoinduction medium (Osteo) or expansion medium (Exp) to produce four microcapsule conditions: Osteo, Osteo+HAP, Exp, and Exp+HAP. Microcapsules facilitated alkaline phosphatase secretion and deposition of bone specific proteins (osteocalcin and osteopontin) by encapsulated MSCs over 28 days of osteogenic culture. SEM and micro-CT analysis showed cell-deposited mineral covering the surfaces of the HAP microgranules and interior of the microcapsule membrane. The mineralized microcapsules could be combined and fused into cylindrical constructs (4 × 5 mm, W × H), and uniaxial compression tests confirmed that microcapsule mineralization greatly enhanced the yield stresses of Osteo and Osteo+HAP fused constructs (10.4 ± 4.4 MPa and 6.4 ± 2.8 MPa), compared to only HAP microgranules (Exp+HAP, 0.5 ± 0.3 MPa). The C4S/CMC/Chitosan microcapsules provide a platform allowing pre-mineralization of microcapsules in vitro for later assembly of larger load-bearing constructs, or for use as an injectable bone regeneration strategy. Statement of SignificanceClinical translation of bone tissue engineering is limited by the difficulty of generating space filling implants that both resist compressive loading, and simultaneously deliver cells throughout the bone defect. Here, we present the design of a microcapsule system containing both stem cells capable of rebuilding bone tissue, and a mechanically tough bone-like mineral, that imparts compression resistance to the microcapsules. The microcapsules support stem cell differentiation to an osteogenic phenotype, that can mineralize the microcapsule membrane and interior. The mineralized microcapsules can be assembled into larger bone constructs, and have mechanical properties on par with trabecular bone.

Autologous oxygen release nano bionic scaffold composite miR-106a induced BMSCs enhances osteoblast conversion and promotes bone repair through regulating BMP-2.

Suitable seed cells and selection of bioactive scaffold materials are the main research contents of bone tissue engineering. It was showed that autologous oxygen release nano bionic scaffold could promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The role of microRNA-106a (miR-106a) in regulating BMSCs differentiation has not been reported. We intend to investigate the role of autologous oxygen release nano bionic scaffold composite miR-106a in inducing BMSCs constructing tissue engineering bone. Rat BMSCs were isolated and transfected by using miR-106a scramble or miR-106a inhibitor. Healthy male Sprague-Dawly (SD) rats were randomly divided into three groups, including bone fracture group established as rat tibial fracture model, negative control group implanted by autologous oxygen release nano bionic scaffold composite miR-106a scramble BMSCs, and miR-106a inhibitor group implanted by autologous oxygen release nano bionic scaffold composite miR-106a inhibitor BMSCs. Callus growth was observed. Alkaline phosphatase (ALP) activity was detected. Bone morphogenetic protein 2 (BMP-2) expression was tested by Real-time PCR (RT-PCR) and Western blot assay. Collagen II production was determined by RT-PCR. Autologous oxygen release nano bionic scaffold composite BMSCs significantly increased local bone mineral density, promoted callus healing, facilitated ALP secretion, elevated collagen II expression, and up-regulated BMP-2 mRNA and protein levels compared with fracture group (p<0.05). Autologous oxygen release nano bionic scaffold composite miR-106a induced BMSCs exhibited more significant effect on bone repair (p<0.05). Autologous oxygen release nano bionic scaffold composite miR-106a induced BMSCs enhanced osteoblast conversion and promoted bone repair through regulating BMP-2.

Protective effects of Tat-DJ-1 protein against streptozotocin-induced diabetes in a mice model.

A major feature of type 1 diabetes mellitus (T1DM) is hyperglycemia and dysfunction of pancreatic β-cells. In a previous study, we have shown that Tat-DJ-1 protein inhibits pancreatic RINm5F β-cell death caused by oxidative stress. In this study, we examined effects of Tat-DJ-1 protein on streptozotocin (STZ)-induced diabetic mice. Wild type (WT) Tat-DJ-1 protein transduced into pancreas where it markedly inhibited pancreatic β-cell destruction and regulated levels of serum parameters including insulin, alkaline phosphatase (ALP), and free fatty acid (FFA) secretion. In addition, transduced WT Tat-DJ-1 protein significantly inhibited the activation of NF-κB and MAPK (ERK and p38) expression as well as expression of COX-2 and iNOS in STZ exposed pancreas. In contrast, treatment with C106A mutant Tat-DJ-1 protein showed no protective effects. Collectively, our results indicate that WT Tat-DJ-1 protein can significantly ameliorate pancreatic tissues in STZ-induced diabetes in mice.

Scutellarin enhances osteoblast proliferation and function via NF-κB-mediated CXCR4 induction

In the current study, we aimed to examine the function of scutellarin on human osteoblast proliferation and osteogenic function. The results indicated that scutellarin enhanced osteoblast proliferation over a seven day period. This increase in cell proliferation was associated with corresponding increases in osteoblast activity, as measured by alkaline phosphatase (ALP) secretion, intracellular calcium ion influx, and calcium deposition. These anabolic effects were associated with C-X-C chemokine receptor type 4 (CXCR4) mRNA levels and protein induction. Knockdown of CXCR4 reversed the scutellarin-induced increases in cell proliferation, ALP activity, and calcium deposition. Furthermore, scutellarin increased p65 phosphorylation in a dose-dependent manner, which resulted in the increased binding of phosphorylated p65 to the CXCR4 gene promoter region, to increase CXCR4 protein expression. p65 phosphorylation inhibition resulted in a decrease in CXCR4 protein expression. A p65 inhibitor blocked scutellarin-induced increases in osteoblast proliferation and function. Moreover, in a rat model of estrogen-deficient osteoporosis, scutellarin restored ovariectomy-induced bone loss in mice. Taken together, both cellular and animal models support the novel findings that scutellarin increased osteoblast proliferation and function through NF-κB/p65-mediated CXCR4 induction.

The effects of strontium incorporation on a novel gelatin/bioactive glass bone graft: In vitro and in vivo characterization

Growing evidence indicates that biomaterials that contain bioactive glass (BG) exert positive effects on bone tissue repair. Strontium-substituted BGs have been demonstrated to promote bone formation while inhibiting bone resorption by osteoclasts. In this study, we fabricated composites containing gelatin combined with different concentrations of strontium (Sr)-delivering glasses. We then investigated the in vitro effect of the presence of Sr in the polymer matrix on proliferation and differentiation of bone marrow mesenchymal stem cells (BMMSCs). The potential angiogenic effect of Sr was assessed by subcutaneous implantation in mice. Scanning electron microscopy (SEM) of prepared scaffolds showed an interconnected porous structure with an average diameter of 100–300 µm; increasing the percentage of Sr decreased the average pore size. The stiffness of scaffolds containing Sr was about five times greater than that of scaffolds containing BG with no Sr. Cytocompatibility of the prepared scaffolds, assessed using BMMSCs, was confirmed with MTT assay. Secretion of alkaline phosphatase was significantly enhanced in cells cultured on Sr-containing scaffolds, compared with other samples, after 14 days. Imunohistofluorescence analysis indicated that the addition of Sr increased perivascular localization and cell migration, and stimulated sprouting angiogenesis. The results of this study highlight the enhanced mechanical and biological properties of our polymer matrix after the addition of Sr. Our results indicate that Sr-containing bioactive glasses could exert beneficial effects on bone tissue engineering.

Composite PLA/PEG/nHA/Dexamethasone Scaffold Prepared by 3D Printing for Bone Regeneration

3D printing has become an essential part of bone tissue engineering and attracts great attention for the fabrication of bioactive scaffolds. Combining this rapid manufacturing technique with chemical precipitation, biodegradable 3D scaffold composed of polymer matrix (polylactic acid and polyethylene glycol), ceramics (nano hydroxyapatite), and drugs (dexamethasone (Dex)) is prepared. Results of water contact angle, differential scanning calorimeter, and mechanical tests confirm that incorporation of Dex leads to significantly improved wettability, higher crystallinity degree, and tunable degradation rates. In vitro experiment with mouse MC3T3-E1 cells implies that Dex released from scaffolds is not beneficial for early cell proliferation, but it improves late alkaline phosphatase secretion and mineralization significantly. Anti-inflammation assay of murine RAW 264.7 cells proves that Dex released from all the scaffolds successfully suppresses lipopolysaccharide induced interleukin-6 and inducible nitric oxide synthase secretion by M1 macrophages. Further in vivo experiment on rat calvarial defects indicates that scaffolds containing Dex promote osteoinduction and osteogenic response and would be promising candidates for clinical applications.

Nutrient Utilization Strategies of Algae and Bacteria after the Termination of Nutrient Amendment with Different Phosphorus Dosage: A Mesocosm Case

ABSTRACTThe impacts of nutrient amendment termination on the growth strategies of algae and bacteria were conducted in experimentally designed mesocosm in which two different phosphorus (P) dosages were treated. The algal community composition did not change greatly in Group A (low phosphorus) and Group B (high phosphorus). In Group A, the secretion of bacterial alkaline phosphatase (AP) after nutrient termination stimulated bacterial phosphorus acquisition, which caused the decrease in algal phosphorus levels, in terms of the increase of bacterial abundance and bacterial production, as well as the decrease in chlorophyll a and particulate organic carbon. The algal collapse resulted in dissolved organic carbon secretion, further fuelling bacterial growth. In Group B, excess phosphorus input urged algae to store phosphorus as poly-phosphate. When phosphorus input ceased, in order to maintain their used high phosphorus demand, algae strengthened to gain phosphorus through the hydrolysis of dissolved organic phosphorus in water column and ploy-phosphate inside the cells by AP, evidenced by high algal alkaline phosphatase activity, algal growth continuation, and bacterial growth decline. These facts indicated that phosphorus content should reduce to a lower level than expected, so that algal bloom can be effectively controlled in eutrophic water bodies.

Microgrooved-surface topography enhances cellular division and proliferation of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells’ (MSCs) fate is largely determined by the various topographical features and a range of extracellular matrix (ECM) components present in their niches. Apart from maintaining structural stability, they regulate cell morphology, division, proliferation, migration and differentiation among others. Traditional MSC cultures, which are mainly based on two-dimensional smooth surfaces of culture dishes and plates, do not provide topographical cues similar to in vivo three-dimensional niches, impacting various cellular processes. Therefore, we culture the mouse bone marrow-derived MSCs on microgrooved bearing surface, partially mimicking in vivo reticulated niche, to study its effect on morphology, pluripotency factor-associated stemness, cell division and rate of proliferation. Following culture, morphological features, and MSC-specific marker gene expression, such as CD29, CD44, Sca-1 along with HSC (Haematopoietic stem cell)-specific markers like CD34, CD45, CD11b were evaluated by microscopy and immunophenotyping, respectively. HSC is another type of bone marrow stem cell population, which concertedly interacts with MSC during various functions, including haematopoiesis. In addition, mesenchymal stem cells were further analyzed for gene expression of pluripotency-associated transcription factors such as Oct3/4, Sox-2, Nanog and Myc, as well as differentiated into adipocytes, osteocytes and chondrocytes. Our results show that microgrooved surface-cultured mesenchymal stem cells (MMSCs) expressed higher levels of expected cell surface and pluripotency-associated markers and proliferated more rapidly (2–3×fold) with higher percentage of cells in S/G2-M-phase, consequently giving rise to higher cell yield compared to standard culture flask-grown cells (MSCs), taken as control. Furthermore, both MSCs and MMSCs showed considerable accumulation of intracellular lipid-droplets, higher alkaline phosphatase activity and secretion of extracellular matrix that are characteristics of adipogenesis, osteogenesis and chondrogenesis, respectively.

A graded graphene oxide-hydroxyapatite/silk fibroin biomimetic scaffold for bone tissue engineering

To better mimic natural bone, a graphene oxide-hydroxyapatite/silk fibroin (cGO-HA/SF) scaffold was fabricated by biomineralizing carboxylated GO sheets, blending with SF, and freeze-drying. The material has increasing porosity and decreasing density from outside to inside. Analysis of GO mineralization in simulated body fluid indicated that carboxylation and Chitosan may synergistically regulate HA growth along the c-axis of weakly crystalline, rod-like GO-HA particles. Compared with HA/SF gradient composites, a cGO-HA gradient scaffold with cGO:HA mass ratio 1:4 has 5-fold and 2.5-fold higher compressive strength and compressive modulus, respectively. Additionally, the cGO-HA/SF composite stimulated mouse mesenchymal stem cell adhesion and proliferation, alkaline phosphatase secretion, and mineral deposition more strongly than HA/SF and pure HA scaffolds. Hence, the material may prove to be an excellent and versatile scaffold for bone tissue engineering.

Insulin-like growth factor-1 promotes osteogenic differentiation and collagen I alpha 2 synthesis via induction of mRNA-binding protein LARP6 expression.

This study explored the mechanism underlying the stimulation of collagen synthesis and osteoblastic differentiation by insulin-like growth factor 1 (IGF1) in primary mouse osteoblasts. Primary mouse calvarial osteoblasts were cultured and treated with various doses of IGF1 before transfection with siRNA targeting the collagen type I alpha 2 (Col1a2) or La ribonucleoprotein domain family member 6 (Larp6) genes. Alkaline phosphatase (ALP) activity, osteocalcin staining, alizarin red quantification and the expression level of runt-related transcription factor 2 (RUNX2) were performed to assess the differentiation of pre-osteoblasts. Based on Western blot analysis, IGF1 up-regulated COL1A2 protein expression in the primary osteoblasts in a dose- and time-dependent manner. In addition, Col1a2 interference inhibited the differentiation and mineralization of osteoblasts. IGF1 also stimulated the differentiation of mouse primary osteoblasts and increased LARP6 expression during osteogenic differentiation. RNA-Immunoprecipitation (IP) indicated that LARP6 could bind to Col1a2 mRNA after IGF1 stimulation. However, transfection of Larp6-specific siRNA significantly reduced collagen and ALP secretion, mineralization and inhibited the expression of osteocalcin and RUNX2, indicating that Larp6 interference inhibited the differentiation ability of primary mouse calvarial osteoblasts, and these effects could not be reversed by IGF1. Thus, IGF1 could promote COL1A2 expression and osteoblast differentiation in primary mouse calvarial pre-osteoblasts by increasing LARP6 expression via a posttranscriptional mechanism.

Calcium Channel Opening Rather than the Release of ATP Causes the Apoptosis of Osteoblasts Induced by Overloaded Mechanical Stimulation

Background: Stress fracture is one of the most common overuse injuries in athletes. Overloaded mechanical stimulation is an important factor affecting stress fractures, but the mechanism is unclear. Methods: MC3T3-E1 cells and a polycaprolactone (PCL) scaffold were co-cultured, and finite element analysis (FEA) was used to analyze the load-carrying capability. Cell proliferation was investigated with CCK-8 assays. An alkaline phosphatase (AKP) activity assay was used to evaluate cell differentiation. Cell apoptosis was analyzed using Hoechst/ PI double-labeling, Caspase-3 activity and lactate dehydrogenase (LDH) activity assays. Realtime PCR and Western blotting were used to examine the gene and protein expression, respectively, of Caspase-3 and Caspase-9. Assays of the intracellular calcium with fluorescent probe technique and extracellular ATP with fluorometric assay kit were used to analyze the changes in the intracellular calcium concentration induced by calcium channel opening and the release of ATP, respectively, at different operation times. Results: When the apparent strain reached 10000 µε, the strain scope of fber at levels greater than 4000 µε was 60%. Overloading for 4 days and operation times of 0.5 h and 2 h increased the cell number and AKP secretion. However, apoptosis genes were activated at the same time, and the operation time of 2 h had a significantly greater effect than 0.5 h. At 8 days, the cell numbers were greater for the operation time of 0.5 h than for 2 h, and the 2-h groups had the fastest apoptosis rate. Overloading for 1 day increased intracellular calcium levels and ATP release. The increase in intracellular calcium could be blocked by the addition of N-ethylmaleimide (NEM) or Hank's medium. Overloading for 8 days increased intracellular calcium levels but decreased extracellular ATP, and verapamil blocked the increase in intracellular calcium. Conclusion: We found that a simultaneous ‘double effect’ on osteoblasts was induced by overloading, which promoted cell proliferation, differentiation and apoptosis. Short-term overloading could open the cell membrane calcium channels and release calcium stores to elevate intracellular calcium levels, thereby promoting the proliferation and differentiation of cells to a greater extent than the effect of apoptosis. For long-term overloading, calcium channel opening in the membrane could lead to overloading of intracellular calcium levels, inducing an apoptosis effect that is greater than the effect on proliferation and differentiation.

Simple additive manufacturing of an osteoconductive ceramic using suspension melt extrusion

ObjectiveCraniofacial bone trauma is a leading reason for surgery at most hospitals. Large pieces of destroyed or resected bone are often replaced with non-resorbable and stock implants, and these are associated with a variety of problems. This paper explores the use of a novel fatty acid/calcium phosphate suspension melt for simple additive manufacturing of ceramic tricalcium phosphate implants. MethodsA wide variety of non-aqueous liquids were tested to determine the formulation of a storable 3D printable tricalcium phosphate suspension ink, and only fatty acid-based inks were found to work. A heated stearic acid-tricalcium phosphate suspension melt was then 3D printed, carbonized and sintered, yielding implants with controllable macroporosities. Their microstructure, compressive strength and chemical purity were analyzed with electron microscopy, mechanical testing and Raman spectroscopy, respectively. Mesenchymal stem cell culture was used to assess their osteoconductivity as defined by collagen deposition, alkaline phosphatase secretion and de-novo mineralization. ResultsAfter a rapid sintering process, the implants retained their pre-sintering shape with open pores. They possessed clinically relevant mechanical strength and were chemically pure. They supported adhesion of mesenchymal stem cells, and these were able to deposit collagen onto the implants, secrete alkaline phosphatase and further mineralize the ceramic. SignificanceThe tricalcium phosphate/fatty acid ink described here and its 3D printing may be sufficiently simple and effective to enable rapid, on-demand and in-hospital fabrication of individualized ceramic implants that allow clinicians to use them for treatment of bone trauma.

In vitro and in vivo study of microporous ceramics using MC3T3 cells, CAM assay and a pig animal model.

Bone tissue engineering combines biomaterials with biologically active factors and cells to hold promise for reconstructing craniofacial defects. In this study the biological activity of biphasic hydroxyapatite ceramics (HA; a bone substitute that is a mixture of hydroxyapatite and β-tricalcium phosphate in fixed ratios) was characterized (1) in vitro by assessing the growth of MC3T3 mouse osteoblast lineage cells, (2) in ovo by using the chick chorioallantoic membrane (CAM) assay and (3) in an in vivo pig animal model. Biocompatibility, bioactivity, bone formation and biomaterial degradation were detected microscopically and by radiology and histology. HA ceramics alone demonstrated great biocompatibility on the CAM as well as bioactivity by increased proliferation and alkaline phosphatase secretion of mouse osteoblasts. The in vivo implantation of HA ceramics with bone marrow mesenchymal stem cells (MMSCs) showed de novo intramembranous bone healing of critical-size bone defects in the right lateral side of pig mandibular bodies after 3 and 9weeks post-implantation. Compared with the HA ceramics without MMSCs, the progress of bone formation was slower with less-developed features. This article highlights the clinical use of microporous biphasic HA ceramics despite the unusually shaped elongated micropores with a high length/width aspect ratio (up to 20) and absence of preferable macropores (>100µm) in bone regenerative medicine.

Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization.

Tissue-engineering scaffolds are often seeded with a single type of cell, but there has been more focus on cocultures to improve angiogenesis and bone formation for craniofacial applications. Investigation of bone-derived osteoblasts (OBs) is important because of the use of bone grafts and migration of OBs from native bone into constructs in vivo and therefore, their contribution to bone formation in vivo. The limitation of primary OBs has been their inability to mineralize without osteogenic factors in vitro. Through coculture of OBs and endothelial cells (ECs) and manipulation of the coculture ratio, mineralization can be achieved without osteogenic media or additional growth factors, thus enhancing their utility for tissue-engineering applications. An optimal ratio of EC/OB for vasculogenesis and mineralization has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios, namely, 10:1, 5:1, 1:1, 1:5, and 1:10 with EC and OB monocultures as controls. The number of vasculogenic networks in a collagen matrix was highest in ratios of 5:1 and 1:1. ECs lined up and formed capillary-like networks by day 10, which was not seen in the other groups. On polystyrene, cells were cocultured with ECs and OBs in direct contact (direct coculture) or separated by a transwell membrane (indirect coculture). At day 21, Alizarin Red staining showed mineralization on the 1:5 and 1:10 direct coculture ratios, with 1:5 having more mineralization nodules present than 1:10. No mineralization was seen in other direct coculture ratios or in any of the indirect coculture ratios. Alkaline phosphatase secretion was highest in the 1:5 direct coculture group. Vascular endothelial growth factor secretion from OBs was present in the 1:5 and 1:10 direct coculture ratios at all time points and inhibited after day 1 in other coculture groups. To improve vasculogenesis, cocultures of primary human ECs and OBs in ratios of 5:1 should be used, but to improve bone formation, the 1:5 direct coculture ratio results in most mineralization.

Total and active microbial communities and phoD as affected by phosphate depletion and pH in soil

Soil microbial communities contribute to organic phosphorus cycling in a variety of ways, including secretion of the PhoD alkaline phosphatase. We sampled a long-term grassland fertilization trial in Switzerland characterized by a natural pH gradient. We examined the effects of phosphate depletion and pH on total and active microbial community structures and on the structure and composition of the total and active phoD-harboring community. Archaeal, bacterial and fungal communities were investigated using T-RFLP and phoD-harboring members of these communities were identified by 454-sequencing. Phosphate depletion decreased total, resin-extractable and organic phosphorus and changed the structure of all active microbial communities, and of the total archaeal and phoD-harboring communities. Organic carbon, nitrogen and phosphorus increased with pH, and the structures of all total and active microbial communities except the total fungal community differed between the two pH levels. phoD-harboring members were affiliated to Actinomycetales, Bacilliales, Gloeobacterales, Planctomycetales and Rhizobiales. Our results suggest that pH and associated soil factors are important determinants of microbial and phoD-harboring community structures. These associated factors include organic carbon and total nitrogen, and to a lesser degree phosphorus status, and active communities are more responsive than total communities. Key players in organic P mineralization are affiliated to phyla that are known to be important in organic matter decomposition.

Regulatory Implications of Non-Trivial Splicing: Isoform 3 of Rab1A Shows Enhanced Basal Activity and Is Not Controlled by Accessory Proteins

Alternative splicing often affects structured and highly conserved regions of proteins, generating so called non-trivial splicing variants of unknown structure and cellular function. The human small G-protein Rab1A is involved in the regulation of the vesicle transfer from the ER to Golgi. A conserved non-trivial splice variant lacks nearly 40% of the sequence of the native Rab1A, including most of the regulatory interaction sites. We show that this variant of Rab1A represents a stable and folded protein, which is still able to bind nucleotides and co-localizes with membranes. Nevertheless, it should be mentioned that compared to other wild-typeRabGTPases, the measured nucleotide binding affinities are dramatically reduced in the variant studied. Furthermore, the Rab1A variant forms hetero-dimers with wild-type Rab1A and its presence in the cell enhances the efficiency of alkaline phosphatase secretion. However, this variant shows no specificity for GXP nucleotides, a constantly enhanced GTP hydrolysis activity and is no longer controlled by GEF or GAP proteins, indicating a new regulatory mechanism for the Rab1A cycle via alternative non-trivial splicing.

Needle-like ion-doped hydroxyapatite crystals influence osteogenic properties of PCL composite scaffolds

Surface topography and chemistry both play a crucial role on influencing cell response in 3D porous scaffolds in terms of osteogenesis. Inorganic materials with peculiar morphology and chemical functionalities may be proficiently used to improve scaffold properties—in the bulk and along pore surface—promoting in vitro and in vivo osseous tissue in-growth.The present study is aimed at investigating how bone regenerative properties of composite scaffolds made of poly(Ɛ-caprolactone) (PCL) can be augmented by the peculiar properties of Mg2+ ion doped hydroxyapatite (dHA) crystals, mainly emphasizing the role of crystal shape on cell activities mediated by microstructural properties. At the first stage, the study of mechanical response by crossing experimental compression tests and theoretical simulation via empirical models, allow recognizing a significant contribution of dHA shape factor on scaffold elastic moduli variation as a function of the relative volume fraction. Secondly, the peculiar needle-like shape of dHA crystals also influences microscopic (i.e. crystallinity, adhesion forces) and macroscopic (i.e. roughness) properties with relevant effects on biological response of the composite scaffold: differential scanning calorimetry (DSC) analyses clearly indicate a reduction of crystallization heat—from 66.75 to 43.05 J g−1—while atomic force microscopy (AFM) ones show a significant increase of roughness—from (78.15 ± 32.71) to (136.13 ± 63.21) nm—and of pull-off forces—from 33.7% to 48.7%. Accordingly, experimental studies with MG-63 osteoblast-like cells show a more efficient in vitro secretion of alkaline phosphatase (ALP) and collagen I and a more copious in vivo formation of new bone trabeculae, thus suggesting a relevant role of dHA to support the main mechanisms involved in bone regeneration.

Preparation of the fast setting and degrading Ca–Si–Mg cement with both odontogenesis and angiogenesis differentiation of human periodontal ligament cells

Develop a fast setting and controllable degrading magnesium–calcium silicate cement (Mg–CS) by sol–gel, and establish a mechanism using Mg ions to stimulate human periodontal ligament cells (hPDLs) are two purposes of this study. We have used the diametral tensile strength measurement to obtain the mechanical strength and stability of Mg–CS cement; in addition, the cement degradation properties is realized by measuring the releasing amount of Si and Mg ions in the simulated body fluid. The other cell characteristics of hPDLs, such as proliferation, differentiation and mineralization were examined while hPDLs were cultured on specimen surfaces. This study found out the degradation rate of Mg–CS cements depends on the Mg content in CS. Regarding in vitro bioactivity; the CS cements were covered with abundant clusters of apatite spherulites after immersion of 24h, while less apatite spherulites were formatted on the Mg-rich cement surfaces. In addition, the authors also explored the effects of Mg ions on the odontogenesis and angiogenesis differentiation of hPDLs in comparison with CS cement. The proliferation, alkaline phosphatase, odontogenesis-related genes (DSPP and DMP-1), and angiogenesis-related protein (vWF and ang-1) secretion of hPDLs were significantly stimulated when the Mg content of the specimen was increased. The results in this study suggest that Mg–CS materials with this modified composition could stimulate hPDLs behavior and can be good bioceramics for bone substitutes and hard tissue regeneration applications as they stimulate odontogenesis/angiogenesis.