Synergetic Cues of Bioactive Nanoparticles and Nanofibrous Structure in Bone Scaffolds to Stimulate Osteogenesis and Angiogenesis

Providing a nanotopological physical cue in concert with a bioactive chemical signal within 3D scaffolds, while it being considered a promising approach for bone regeneration, has yet to be explored. Here, we develop 3D porous scaffolds that are networked to be a nanofibrous structure and incorporated with bioactive glass nanoparticles (BGn) to tackle this issue. The presence of BGn and nanofibrous structure (BGn + nanofibrous) substantially increased the surface area, hydro-affinity and protein loading capacity of scaffolds. In particular, the BGn released Si and Ca ions to the levels known to be biologically effective, offering the bone scaffold an ability to deliver therapeutic ions. The mesenchymal stem cells (MSCs) from rats exhibited significantly accelerated adhesion events including cell anchorage, cytoskeletal extensions, and the expression of adhesion signaling molecules on the BGn/nanofibrous scaffolds. The cells gained a more rapid proliferation and migration (penetration) ability over 2 weeks within the BGn + nanofibrous scaffolds than within either nanofibrous or BGn scaffolds. The osteogenesis of MSCs, as confirmed by the expressions of bone-associated genes and proteins, as well as the cellular mineralization was significantly stimulated by the BGn and nanofibrous topology in a synergistic manner. The behaviors of endothelial cells (HUVECs) including cell migration and tubule networking were also enhanced when influenced by the BGn and nanofibrous scaffolds (but more by BGn than by nanofiber). A subcutaneous tissue implantation of the scaffolds further evidenced the in vivo stimulation of neo-blood vessel formation by the BGn + nanofibrous cues, suggesting the possible promising role in bone regeneration. Taken together, the therapeutic ions and nanofibrous topology implemented within 3D scaffolds are considered to play synergistic actions in osteogenesis and angiogenesis, implying the potential usefulness of the BGn + nanofibrous scaffolds for bone tissue engineering

Evaluation of Strontium-Doped Nanobioactive Glass Cement for Dentin–Pulp Complex Regeneration Therapy

Introducing new generations of injectable bioactive types of cement that fulfill excellent injectability, rapid self-setting, high bioactivity, proper biodegradability, and fast therapeutic ion-releasing capability is highly demanded for tooth and bone regeneration. Here, we announce therapeutic fast ion-releasing nanobiocements (NBCs) based on sol–gel-processed calcium silicate mesoporous nanobioactive glass with or without strontium (NBC, Sr-free and Sr-NBC, Sr-doped). The stimulating role of Sr ions in odontogenesis of stem cells derived from dental pulp (DPSCs) and in in vivo dentin formation has been investigated. The nanobiocement was formulated through the mixing of bioactive glass nanopowder with a phosphate-buffered saline (P/L = 0.5 g/mL) to form a soft cement paste that hardens within 5–10 min in the ambient environment. The self-setting originated from a setting reaction involving the deposition of hydroxyapatite as evidenced from X-ray diffraction. Both nanobiocements showed the rapid release of therapeutic ions with biologically effective doses, including strontium (Sr), calcium (Ca), or silicon (Si). In vitro cell cultures with DPSCs showed excellent biocompatibility and high odontogenic potential, especially from Sr-NBC. In an in vivo study, Sr-NBC showed more new dentin formation compared to that of NBC, revealed by two different animal models (odontogenesis in subcutaneous and natural tooth environment). Also, NBCs showed high loading capacities of simvastatin used as a model drug. Taken together, Sr-NBC could be considered as a multifunctional nanobiocement with high bioactivity, excellent biodegradability, fast therapeutic ion release, and high drug loading capability, which potentiates its application in dentin–pulp complex regeneration therapy

Surface-Engineered Hybrid Gelatin Methacryloyl with Nanoceria as Reactive Oxygen Species Responsive Matrixes for Bone Therapeutics

Designing various transplantable biomaterials, especially nanoscale matrixes for bone regeneration, involves precise tuning of topographical features. The cellular fate on such engineered surfaces is highly influenced by many factors imparted by the surface modification (hydrophilicity, stiffness, porosity, roughness, ROS responsiveness). Herein, hybrid matrixes of gelatin methacryloyl (GelMA) decorated with uniform layers of nanoceria (nCe), called Ce@GelMA, were developed without direct incorporation of nCe into the scaffolds. The fabrication involves a simple base-mediated in situ deposition in which uniform nCe coatings were first made on GelMA hydrogels and then nCe layered GelMA scaffolds were made by cryodesiccation. In this hybrid platform, degradable GelMA biopolymer provides the porous microstructure and nCe provides the nanoscaled biointerface. The surface morphology and elemental composition of the matrixes analyzed by field emission scanning electron microscopy (FE-SEM) and energy-dispersive spectroscopy (EDS) show uniform nCe distribution. The surface nanoroughness and chemistry of the matrixes were also characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The presence of nCe on GelMA enhanced its mechanical properties as confirmed by compressive modulus analysis. Substantial bonelike nanoscale hydroxyapatite formation was observed on scaffolds after simulated body fluid (SBF) immersion, which was confirmed by SEM, X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) spectroscopy. Moreover, the developed scaffolds could also be used as an antioxidant matrix owing to the reactive oxygen species (ROS) scavenging property of nCe as assessed by 3,3′,5,5′-tetramethylbenzidine (TMB) assay. The enhanced proliferation and viability of rat bone marrow mesenchymal stem cells (rMSCs) on the scaffold surface after 3 days of culture ensures the biocompatibility of the proposed material. Considering all, it is proposed that the micro/nanoscaled matrix could mimic the composition and function of hard tissues and could be utilized as degradable scaffolds in engineering bones

Characteristics of MBNs before and after amination_.

<p>Transmission electron microscopic images showing MBNs (a) and MBNs-NH₂ (b) with a highly mesoporous structure. (<b>c</b>) X-ray diffraction (XRD) pattern of MBNs-NH₂ showing an amorphous glass phase. (<b>d</b>) Amine functionalization was confirmed on Fourier transform infrared spectroscopy (FTIR). (<b>e</b>) A shift of zeta (ξ)-potential to positive charge was observed after amine functionalization and summary of mesoporous characteristics of MBNs and MBNs-NH₂. (<b>f</b>) XRD pattern of MBNs-NH₂ after immersion in simulated body fluid for 14 or 28 days showing hydroxyapatite precipitation. (<b>g</b>) Cumulative release of ions from MBNs-NH₂ detected by inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis. The measurements were performed in triplicate, and representative results are shown.</p

The purification of bioconjugates of azaphtalocyanines by SPE

Diploma thesis The purification of bioconjugates of azaphthalocyanines by SPE Jakub Cichý Charles Univerzity in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical chemistry and drug analysis This thesis occupy the development of a method for purification biocunjugate azaphthalocyanines by solid-phase extraction (SPE). These derivates of azaphthalocyanine are investigated as molecular probes to guench of fluorescence in various genetic analysis. Experiments were carried out successuvely in order to find the ideal SPE conditions for the analyte. We tried also different type of SPE columns and their influence on the extraction. Subsequently were optimized each extraction steps, the changes in pH, molarity and strength elution solutions were made with an effort to get the best separation results. Column appeared as the best DSC-Ph (500 mg/3 ml) and the most optimal conditions were:  Condition: 3 ml 100% MeOH + 5 ml 50mM TEAA  Apply sample: 100 μl 100nM  Washing solution: 9 ml 55% MeOH/50mM TEAA  Elution solution: 3 ml 80% MeOH/50mM TEAA We obtained by this method recovery of extraction analyte around 70-75 %, with 10% relative contamination of remains oligonucleotide chains in the final eluate solution. The SPE method is an alternative to the already developed HPLC method..

Uptake of MBNs–NH₂ into rDPSCs within 4 h with or without pre-treatment using various types of inhibitors or culture conditions.

<p>Incubation at 4°C was used to prevent ATP-dependent endocytosis. (<b>a</b>) Uptake of FITC-labeled MBNs–NH₂ into rDPSCs was characterized by flow cytometry depending on incubating time and on pre-treatment condition (1 h), which included sodium azide (SA) (100 mM), 5-(N-ethyl-N-isopropyl) amiloride (AR) (2.5 mM), amantadine-HCl (AT) (1 mM), and genistein (GE) (100 mM). Different letters indicate significant differences at p<0.05. Confocal images of rDPSCs incubated with MBNs–NH₂. (<b>b</b>) 3D reconstructions and views of the xz- and yz-planes showing the FITC-labeled MBNs–NH₂ (green) internalized by the cells and associated with the actin cytoskeleton (red). (<b>c—f</b>) Depending on the incubation time, increases in FITC-labeled MBNs–NH₂ (green) were detected in the rDPSCs (Red = F-actin filaments, blue = nucleus, and green = FITC-labeled MBNs–NH₂.) The measurements were performed in triplicate, and representative data or images are shown.</p

Expressions of proteins related with ligamentogenesis of the PDL cells, as analyzed by an ELISA.

<p>(a) Periostin, (b) tenascin, and (c) TGF-β. Results presented when normalized to the static condition with random nanofiber. (^a<i>p</i> < 0.05 compared to DA, ^b<i>p</i> < 0.05 compared to SA, by ANOVA).</p

Illustrative images showing the PDL defect models used in this study.

<p>(a) Photograph of rat premaxillary operation field. Note the dimensions of the defect used to produce standardized 4 mm diameters round full-thickness defects on the lateral surface of premaxilla bone. Two defects were created on one animal and were covered with tissue-engineered construct. (b) Harvested specimens of rat premaxillary operation field after sacrifice. (c) Representative histology image of HE staining of new bone tissue formed within the defect at 4 weeks (black arrow: defect margins) (Magnification x40, scale bar 500 μm). (d) 2D and (e) 3D μCT images. The original outline of the 4 mm defect is clear (white arrow).</p

Micro-CT image analyses results of bone regeneration.

<p>(a) % bone volume (b) bone surface, and (c) bone surface density. The graph represented statistically significant differences among the study groups on the quantification of new bone formation in premaxillary defects after 4 weeks of healing. (^a<i>p</i> < 0.05 compared to DA (remov); ^b<i>p</i> < 0.05 compared to SA (remov); ^c<i>p</i> < 0.05 compared to DA (sound), by ANOVA).</p

Effect of the cyclic uniaxial stretch on the orientation of PDL cells.

<p>Examination of cell shaping on the differently-aligned nanofibers with or without applying dynamic mechanical load. F-actins visualized with rhodamine-phalloidin (red) and nuclei stained with DAPI (cyan) for fluorescent images. Red arrows indicate stretch direction.</p