Efficient in vitro delivery of Noggin siRNA enhances osteoblastogenesis.

Several types of serious bone defects would not heal without invasive clinical intervention. One approach to such defects is to enhance the capacity of bone-formation cells. Exogenous bone morphogenetic proteins (BMP) have been utilized to positively regulate matrix mineralization and osteoblastogenesis, however, numerous adverse effects are associated with this approach. Noggin, a potent antagonist of BMPs, is an ideal candidate to target and decrease the need for supraphysiological doses of BMPs. In the current research we report a novel siRNA-mediated gene knock-down strategy to down-regulate Noggin. We utilized a lipid nanoparticle (LNP) delivery strategy in pre-osteoblastic rat cells. In vitro LNP-siRNA treatment caused inconsequential cell toxicity and transfection was achieved in over 85% of cells. Noggin siRNA treatment successfully down-regulated cellular Noggin protein levels and enhanced BMP signal activity which in turn resulted in significantly increased osteoblast differentiation and extracellular matrix mineralization evidenced by histological assessments. Gene expression analysis showed that targeting Noggin specifically in bone cells would not lead to a compensatory effect from other BMP negative regulators such as Gremlin and Chordin. The results from this study support the notion that novel therapeutics targeting Noggin have the clinically relevant potential to enhance bone formation without the need for toxic doses of exogenous BMPs. Such treatments will undeniably provide safe and economical treatments for individuals whose poor bone repair results in permanent morbidity and disability.

Selective and High Dynamic Range Assay Format for Multiplex Detection of Pathogenic Pseudomonas aeruginosa, Salmonella typhimurium, and Legionella pneumophila RNAs Using Surface Plasmon Resonance Imaging.

Due to its well-characterized and highly conserved structure, as well as its relative abundance in metabolically active cells, bacterial 16S rRNA sequence plays an important role in microbial identification. In this work, a biosensing strategy has been developed for simultaneous detection of 16S rRNA analytes of three pathogenic bacterial strains: Legionella pneumophila, Pseudomonas aeruginosa, and Salmonella typhimurium. Surface plasmon resonance imaging (SPRi) was used as a detection technique coupled with DNA probe sandwich assemblies and gold nanoparticles (GNPs) for signal amplification. The targets 16S rRNA were selectively captured at the interface of the biosensor by surface-bound DNA probes through a hybridization process. GNP-grafted DNA detection probes were then introduced and were hybridized with a defined 16S rRNA region on the long DNA-RNA sandwich assemblies, resulting in a significant increase of the SPR signal. The results demonstrated the successful implementation of this strategy for detecting 16S rRNA sequences in total RNA mixed samples extracted from the three pathogenic strains at a concentration down to 10 pg mL-1 with a large dynamic range of 0.01-100 ng mL-1 and high selectivity. Since no particular optimization of the probe design was applied, this method should be relatively easy to adapt for quantification of a wide range of bacteria in various liquids.

Dielectric spectroscopy platform to measure MCF10A epithelial cell aggregation as a model for spheroidal cell cluster analysis.

3-Dimensional cell cultures are more representative of the native environment than traditional cell cultures on flat substrates. As a result, 3-dimensional cell cultures have emerged as a very valuable model environment to study tumorigenesis, organogenesis and tissue regeneration. Many of these models encompass the formation of cell aggregates, which mimic the architecture of tumor and organ tissue. Dielectric impedance spectroscopy is a non-invasive, label free and real time technique, overcoming the drawbacks of established techniques to monitor cell aggregates. Here we introduce a platform to monitor cell aggregation in a 3-dimensional extracellular matrix using dielectric spectroscopy. The MCF10A breast epithelial cell line serves as a model for cell aggregation. The platform maintains sterile conditions during the multi-day assay while allowing continuous dielectric spectroscopy measurements. The platform geometry optimizes dielectric measurements by concentrating cells within the electrode sensing region. The cells show a characteristic dielectric response to aggregation which corroborates with finite element analysis computer simulations. By fitting the experimental dielectric spectra to the Cole-Cole equation, we demonstrated that the dispersion intensity Δε and the characteristic frequency fc are related to cell aggregate growth. In addition, microscopy can be performed directly on the platform providing information about cell position, density and morphology. This platform could yield many applications for studying the electrophysiological activity of cell aggregates.

Composite biopolymers for bone regeneration enhancement in bony defects.

For the past century, various biomaterials have been used in the treatment of bone defects and fractures. Their role as potential substitutes for human bone grafts increases as donors become scarce. Metals, ceramics and polymers are all materials that confer different advantages to bone scaffold development. For instance, biocompatibility is a highly desirable property for which naturally-derived polymers are renowned. While generally applied separately, the use of biomaterials, in particular natural polymers, is likely to change, as biomaterial research moves towards mixing different types of materials in order to maximize their individual strengths. This review focuses on osteoconductive biocomposite scaffolds which are constructed around natural polymers and their performance at the in vitro/in vivo stages and in clinical trials.

Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy.

Human pancreatic islets are seldom assessed for dynamic responses to external stimuli. Thus, the elucidation of human islet functionality would provide insights into the progression of diabetes mellitus, evaluation of preparations for clinical transplantation, as well as for the development of novel therapeutics. The objective of this study was to develop a microfluidic platform for in vitro islet culture, allowing the multi-parametric investigation of islet response to chemical and biochemical stimuli. This was accomplished through the fabrication and implementation of a microfluidic platform that allowed the perifusion of islet culture while integrating real-time monitoring using impedance spectroscopy, through microfabricated, interdigitated electrodes located along the microchamber arrays. Real-time impedance measurements provide important dielectric parameters, such as cell membrane capacitance and cytoplasmic conductivity, representing proliferation, differentiation, viability, and functionality. The perifusion of varying glucose concentrations and monitoring of the resulting impedance of pancreatic islets were performed as proof-of-concept validation of the lab-on-chip platform. This novel technique to elucidate the underlying mechanisms that dictate islet functionality is presented, providing new information regarding islet function that could improve the evaluation of islet preparations for transplantation. In addition, it will lead to a better understanding of fundamental diabetes-related islet dysfunction and the development of therapeutics through evaluation of potential drug effects.

Dielectric spectroscopy for monitoring human pancreatic islet differentiation within cell-seeded scaffolds in a perfusion bioreactor system.

The long-term in vitro culture and differentiation of human pancreatic islets is still hindered by the inability to emulate a suitable microenvironment mimicking physiological extracellular matrix (ECM) support and nutrient/oxygen perfusion. This is further amplified by the current lack of a non-invasive and rapid monitoring system to readily evaluate cellular processes. In this study, we realized a viable method for non-invasively monitoring isolated human pancreatic islets in vitro. Islets are induced to dedifferentiate into proliferative duct-like structures (DLS) in preparation for potential and subsequent re-differentiation into functional islet-like structures (ILS) in a process reminiscent of islet regeneration strategies. This long-term in vitro process is conducted within a three-dimensional microenvironment involving islets embedded in an optimized ECM gel supported by microfabricated three-dimensional scaffolds. The islet-scaffold is then housed and continuously perfused within chambers of a bioreactor platform. The process in its entirety is monitored through dielectric spectroscopy measurements, yielding an accurate representation of cellular morphology, functionality, and volume fraction. This non-invasive and real-time monitoring tool can be further manipulated to elucidate important information about the optimized cellular microenvironment required for maintaining long-term culture and achieve efficient differentiation for islet regeneration.

Investigation of probiotic bacteria as dental caries and periodontal disease biotherapeutics.

Oral diseases, specifically dental caries and periodontal disease, are characterised by increases in pathogenic microorganisms, increased demineralisation and increased inflammation and levels of inflammatory markers. Despite the therapeutic strategies, oral diseases have elevated prevalence rates. Recent work has demonstrated that probiotic bio-therapeutics can decrease oral pathogen counts, including caries-causing Streptococcus mutans and oral inflammation. The aim of this work was to investigate putative probiotic bacteria, selected for S. mutans inhibition and for their oral health-promoting characteristics. The probiotic bacteria were screened for S. mutans inhibition, probiotic bacteriocin activity, salivary pH modulation, probiotic nutrient (sucrose) competition, probiotic co-aggregation with S. mutans, bacterial attachment to oral epithelial keratinocytes, bacterial nitric oxide production and bacterial antioxidant activity. The results indicate that Lactobacillus reuteri strains NCIMB 701359, NCIMB 701089, NCIMB 702655 and NCIMB 702656 inhibited S. mutans to non-detectable levels (<10 cfu/ml). L. reuteri strains also demonstrated the highest antioxidant capacity of the tested strains (7.73-13.99 µM Trolox equivalents), suggesting their use as both caries and periodontal disease therapeutics. Although Lactobacillus fermentum NCIMB 5221 inhibited S. mutans at lower levels, it significantly buffered the pH (4.18) of saliva containing S. mutans, co-aggregated with S. mutans (10.09%), demonstrated high levels of sucrose consumption (138.11 mM) and successfully attached to gingival epithelial cells (11%). This study identified four L. reuteri strains and one L. fermentum strain to be further investigated as oral disease biotherapeutics.

Characterization and biocompatibility studies of new degradable poly(urea)urethanes prepared with arginine, glycine or aspartic acid as chain extenders.

Polyurethanes are very often used in the cardiovascular field due to their tunable physicochemical properties and acceptable hemocompatibility although they suffer from poor endothelialization. With this in mind, we proposed the synthesis of a family of degradable segmented poly(urea)urethanes (SPUUs) using amino acids (L-arginine, glycine and L-aspartic acid) as chain extenders. These polymers degraded slowly in PBS (pH 7.4) after 24 weeks via a gradual decrease in molecular weight. In contrast, accelerated degradation showed higher mass loss under acidic, alkaline and oxidative media. MTT tests on polyurethanes with L-arginine as chain extenders showed no adverse effect on the metabolism of human umbilical vein endothelial cells (HUVECs) indicating the leachables did not provoke any toxic responses. In addition, SPUUs containing L-arginine promoted higher levels of HUVECs adhesion, spreading and viability after 7 days compared to the commonly used Tecoflex(®) polyurethane. The biodegradability and HUVEC proliferation on L-arginine-based SPUUs suggests that they can be used in the design of vascular grafts for tissue engineering.

Platelet adhesion and human umbilical vein endothelial cell cytocompatibility of biodegradable segmented polyurethanes prepared with 4,4'-methylene bis(cyclohexyl isocyanate), poly(caprolactone) diol and butanediol or dithioerythritol as chain extenders.

Biodegradable segmented polyurethanes were prepared with poly(caprolactone) diol as a soft segment, 4,4'-methylene bis(cyclohexyl isocyanate) (HMDI) and either butanediol or dithioerythritol as chain extenders. Platelet adhesion was similar in all segmented polyurethanes studied and not different from Tecoflex® although an early stage of activation was observed on biodegradable segmented polyurethane prepared with dithioerythritol. Relative viability was higher than 80% on human umbilical vein endothelial cells in contact with biodegradable segmented polyurethane extracts after 1, 2 and 7 days. Furthermore, both biodegradable segmented polyurethane materials supported human umbilical vein endothelial cell adhesion, spreading, and viability similar to Tecoflex® medical-grade polyurethane. These biodegradable segmented polyurethanes represent promising materials for cardiovascular applications.

Enhanced SPR response from patterned immobilization of surface bioreceptors on nano-gratings.

In this report, nano-gratings with guided adsorption of biomolecules are investigated as new transducer elements or biointerfaces for surface plasmon resonance biosensor technologies. SPR biosensors are of particular interest due to the interaction between the electromagnetic fields and periodic nano-structures. In this article, sensitivity enhancement is demonstrated for a surface plasmon resonance interface, in a Kretschmann's configuration, featuring nano-gratings combined with nano-patterned immobilization of surface bioreceptors. The fabrication of this enhanced biointerface is demonstrated using a combination of metal lift-off and self-assembled monolayers. Rigorous coupled-wave analyses point to an increase in SPR angular response for the immobilization of surface bioreceptors onto areas of the nano-corrugated surface exhibiting high electromagnetic field intensity. Experimental measurements of the immobilization of anti-TNF-alpha antibody as a model bioreceptor using an imaging-SPR technique show a 3 times increase in angular resonance response from nano-grating surfaces with functionalized mesas compared to a planar surface or to a uniformly functionalized nano-grating surface. Furthermore, results also show an increased detection of TNF-alpha due to the increased accessibility to the adsorbed bioreceptors on the nano-gratings.

Fabrication and characterization of patterned immobilization of quantum dots on metallic nano-gratings.

Surfaces featuring nano-structures and biochemical patterns are increasingly developed as novel and superior substrates for biosensors and assays. Metallic periodic nano-structures have been studied for their unique optical properties and in particular their ability to support surface plasmon waves. Here we present a new nano-structuring approach based on gentle metal lift-off process coupled with self-assembled surface chemistry for the fabrication of a zeroth-order 400nm period metallic grating with differentiated surface chemistries on the mesas and troughs. The approach, using terminated self-assembled monolayers, creates versatile functionalized substrates allowing the precise deposition of complex biomolecular structures. We use this technique to perform the guided deposition of a three-dimensional polyelectrolyte multilayer structure and the patterned adsorption of quantum dots. Finally, we demonstrate that scanning near-field optical microscopy, used in conjuncture with atomic force microscopy and scanning electron microscopy, is an ideal tool for the characterization of this nano-structured surface as it provides a complete chemical, topographical and optical image of the surface. This ability to pattern and locally measure the surface properties is likely to have an important impact on the design of novel and optimized biointerfaces and transducers for biosensors.

The influence of isocyanurate content on the bioperformance of hydrocarbon-based polyurethanes.

Bulk, surface and bioactivity of newly synthesized hydroxy telechelic polyisoprene-based (H-HTPI) polyurethane were investigated by means of ATR-FT-IR, contact-angle measurements, cell viability, calcification, and platelet and fibrinogen quantification. The influence of isophorone diisocyanates isocyanurate (I-IPDI) content on these properties was determined. Results generally showed a non-significant difference in these properties when they were compared with a commercially available biomedical polyurethane (PU), such as Tecoflex. Unexpectedly, where the increase of isocyanate content for commercial diisocyanate-based biocompatible PU significantly increases the surface contact angle, the new hydroxy telechelic polyisoprene-based PU showed a decrease of water contact angle with increasing I-IPDI content in the polymer. Nevertheless, the overall surface exhibited hydrophobic properties, i.e., theta > 85. Polymer cytotoxicity, assessed with L929 cell line in direct contact with the surface of the samples, showed no toxic effects on the cells. Interestingly, regardless of the I-IPDI content, platelet adhesion and fibrinogen adsorption, as well as the mineral deposition were fairly similar for all synthesized PUs. Our findings revealed that replacing diisocyanates by their isocyanurate homologues is a very relevant approach for preparation of polyurethanes with different mechanical properties while maintaining similar surface properties.

Enhanced surface plasmon resonance imaging detection of DNA hybridization on periodic gold nanoposts.

We explore periodic gold nanoposts as substrates for the enhanced surface plasmon resonance imaging (SPRi) detection of DNA hybridization. Rigorous coupled-wave analysis was used to model and design the nanopost-based SPRi biosensor. Arrayed gold nanoposts on gold-coated glass substrate, with various widths and periodicity, were fabricated using electron-beam lithography and characterized with scanning electron and atomic force microscopy. A scanning-angle SPRi apparatus was used to conduct the kinetic analysis of DNA hybridization on nanopost-based sensor surface and assess the corresponding SPR signal amplification. Experimental results showed that both the nanostructure size and period influenced the SPR signal enhancement; the optimized 30 nm height, 50 nm size, and 110 nm period nanoposts provided a fivefold SPR signal amplification compared with the plain 50 nm thick gold film used as control.

Nondestructive online in vitro monitoring of pre-osteoblast cell proliferation within microporous polymer scaffolds.

We present a system for the online, in vitro, nondestructive monitoring of tissue growth within microporous polymer scaffolds. The system is based on measuring the admittance of the sample over a frequency range of 10-200 MHz using an open-ended coaxial probe and impedance analyzer. The sample admittance is related to the sample complex permittivity (CP) by a quasi-static model of the probe's aperture admittance. A modified effective medium approximation is then used to relate the CP to the cell volume fraction. The change of cell volume fraction is used as a measure of tissue growth inside the scaffold. The system detected relative cell concentration differences between microporous polymer scaffolds seeded with 0.4, 0.45, 0.5, and 0.6 x 10(6) pre-osteoblast cells. In addition, the pre-osteoblast proliferation within 56 scaffolds over 14 days was recorded by the system and a concurrent DNA assay. Both techniques produced cell proliferation curves that corresponded to those found in literature. Thus, our data confirmed that the new system can assess relative cell concentration differences in microporous scaffolds enabling online nondestructive tissue growth monitoring.

Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress.

The use of surface plasmon resonance (SPR) biosensors is increasingly popular in fundamental biological studies, health science research, drug discovery, clinical diagnosis, and environmental and agricultural monitoring. SPR allows for the qualitative and quantitative measurements of biomolecular interactions in real-time without requiring a labeling procedure. Today, the development of SPR is geared toward the design of compact, low-cost, and sensitive biosensors. Rapid advances in micro-fabrication technology have made available integratable opto-electronic components suitable for SPR. This review paper focuses on the progress made over the past 4 years toward this integration. Readers will find the descriptions of novel SPR optical approaches and materials. Nano-technology is also increasingly used in the design of biologically optimized and optically enhanced surfaces for SPR. Much of this work is leading to the integration of sensitive SPR to lab-on-a-chip platforms.

Enzymatically-generated fluorescent detection in micro-channels with internal magnetic mixing for the development of parallel microfluidic ELISA.

The Enzyme-Linked Immuno-Sorbent Assay, or ELISA, is commonly utilized to quantify small concentrations of specific proteins for a large variety of purposes, ranging from medical diagnosis to environmental analysis and food safety. However, this technique requires large volumes of costly reagents and long incubation periods. The use of microfluidics permits one to specifically address these drawbacks by decreasing both the volume and the distance of diffusion inside the micro-channels. Existing microfluidic systems are limited by the necessary control of extremely low flow rates to provide sufficient time for the molecules to interact with each other by diffusion only. In this paper, we describe a new microfluidic design for the realization of parallel ELISA in stop-flow conditions. Magnetic beads were used both as a solid phase to support the formation of the reactive immune complex and to achieve a magnetic mixing inside the channels. In order to test the detection procedure, the formation of the immune complex was performed off-chip before the reactive beads were injected into the reaction chamber. Anti-streptavidin antibodies were quantified with low picomolar sensitivity (0.1-6.7 pM), a linear range of 2 orders of magnitude and good reproducibility. This work represents the first step toward a new platform for simple, highly effective and parallel microfluidic ELISA.

In vitro thrombogenicity investigation of new water-dispersible polyurethane anionomers bearing carboxylate groups.

New segmented polyurethane (PU) anionomers based on hydroxytelechelic polybutadiene were synthesized via an aqueous dispersion process. Incorporation of carboxylic groups was achieved using thioacids of different length. Surface properties were investigated by mean of water absorption analysis and static contact-angle measurements using water, diiodomethane, formamide and ethylene glycol. Blood compatibility of the PUs was evaluated by in vitro adhesion assays using 111In-radiolabeled platelet-rich plasma and [125I]fibrinogen. Morphology of the adhered platelets was examined by scanning electron microscopy (SEM). Results were compared to two biomedical-grade PUs, namely Pellethane and Tecoflex. Insertion of carboxylic groups increased surface hydrophilicity and limited water uptake ( < 8% for an ion content of 5% by weight). Surface energy of all synthesized PUs was between 40 and 45 mJ/m2. Platelet adhesion and fibrinogen adsorption on the PU anionomer surfaces were affected as a function to the increase of graft length; thiopropionic was the most haemocompatible, followed by thiosuccinic and then thioglycolic acid. SEM analyses of all ionic PU samples exhibited low platelet adhesion to surfaces with no morphological modification. In conclusion, increased hydrophily, dynamic mobility and charge repulsion are synergistic key factors for enhanced haemocompatibility.

A Highly Responsive System for On-line in vitro Assessment of Tissue Growth within MicroPorous Polymer Scaffolds.

We have proposed a highly responsive system for the on-line in vitro assessment of tissue growth within microporous polymer scaffolds that obviates any compromise of sample integrity. The system's function is based on the sample's loss factor: the imaginary part of the complex permittivity. Reflection measurements were performed using an open-ended coaxial probe and impedance analyzer; they were then related to the sample's complex permittivity by a quasi-static model of the probe's aperture admittance. Measurements of saline solutions showed that the real part of permittivity was corrupted by apparent polarization effects. Consequently, we developed a simplified formulation of the imaginary part of the Hanai-Wagner effective medium approximation to eliminate its dependence on the real part of complex permittivity measurement. This formulation allows the sample's cell concentration to be determined. The variation of a sample's cell concentration over time was used as a measure of tissue growth. Measurements in the frequency range of 10-200 MHz were performed on micro-porous polymer scaffolds seeded with progressively greater number of cells. Results demonstrated that the system detected concentration differences between cell-seeded scaffolds.

Hemocompatibilty of new ionic polyurethanes: influence of carboxylic group insertion modes.

New segmented polyurethane (PU) anionomers based on hydroxytelechelic polybutadiene (HTPB) were synthesized via two environment-friendly chemical routes. The effects of carboxylic content and ion incorporation mode on the surface properties were investigated by mean of water absorption analysis and static contact angle measurements using water, diiodomethane, formamide and ethylene glycol. Blood compatibility of the PUs was evaluated by in vitro adhesion assay using 111In-radiolabeled platelet rich plasma and 125I-fibrinogen. The morphology of platelet adhesion was also observed by scanning electron microscopy (SEM). Results were compared with a biomedical-grade PU, Pellethane. Insertion of the carboxylic groups on the soft segments (S-alpha series), using thioglycolic acid (TGA), increases surface hydrophilicity, limits water uptake (5%, for an ion content of 3.6 wt%), and reduces platelet adhesion and fibrinogen adsorption on the PUs' surfaces. In contrast, the classical insertion onto the hard segment (H-alpha series), using dimethylolpropionate (DMPA) as chain extender, leads to high water uptake (18%, for an ion content of 3.6 wt%) and promotes platelet and fibrinogen adhesion. SEM analyses of the non-ionic PUs exhibited surfaces with adhered platelets which underwent morphological modification. Similarly, the H-alpha ionic PUs show adherent and activated platelets. On the contrary, no platelet morphology changes were observed on the S-alpha ionic surfaces. In conclusion, insertion of carboxyl groups on the soft segments of PUs reduces their thrombogenicity.

Nitinol versus stainless steel stents: acute thrombogenicity study in an ex vivo porcine model.

Acute and subacute stents thrombosis along with thrombus mediating neointimal proliferation within the stent struts remain major concerns in coronary stenting. Up to date, there is an obvious lack of data on the thrombogenicity of stent materials in physiological conditions. This study was performed to compare the relative thrombogenicity of nitinol versus stainless steel stents. Nitinol stents were laser cut to reproduce the exact geometry of the stainless steel Palmaz stents and tested in an ex vivo AV shunt porcine model under controlled conditions. Nitinol stents presented only small amounts of white and/or red thrombus principally located at the strut intersections while Palmaz stents clearly exhibited more thrombus. As a result, 125I-fibrin(ogen) adsorption and (111)I-platelets adhesion were significantly lower on nitinol than on stainless steel devices (36%, p = 0.03 for fibrin(ogen) and 63%, p = 0.01 for platelet). These results were confirmed by scanning electron observations showing different thrombus morphologies for nitinol and stainless steel. Along with the unique mechanical properties of nitinol, its promising haemocompatibility demonstrated in our study may promote their increasing use for both peripheral and coronary revascularization procedures.