Diboronic-Acid-Based Electrochemical Sensor for Enzyme-Free Selective and Sensitive Glucose Detection.

A diboronic acid anthracene-based fluorescent system for detecting blood glucose could be used for 180 days. However, there has not yet been a boronic acid immobilized electrode to selectively detect glucose in a signal-increased way. Considering malfunctions of sensors at high sugar levels, the electrochemical signal should be increased proportionally to the glucose concentration. Therefore, we synthesized a new diboronic acid derivative and fabricated the derivative-immobilized electrodes for the selective detection of glucose. We performed cyclic voltammetry and electrochemical impedance spectroscopy with an Fe(CN)63-/4- redox pair for detecting glucose in the range of 0-500 mg/dL. The analysis revealed increased electron-transfer kinetics such as increased peak current and decreased semicircle radius of Nyquist plots as the glucose concentration increased. The cyclic voltammetry and impedance spectroscopy showed that the linear detection range of glucose was 40 to 500 mg/dL with limits of detection of 31.2 mg/dL and 21.5 mg/dL, respectively. We applied the fabricated electrode to detect glucose in artificial sweat and obtained 90% of the performance of the electrodes in PBS. Cyclic voltammetry measurements of other sugars such as galactose, fructose, and mannitol also showed linear increased peak currents proportional to the concentrations of the tested sugars. However, the slopes of the sugars were lower than that of glucose, indicating selectivity for glucose. These results proved the newly synthesized diboronic acid is a promising synthetic receptor for developing a long-term usable electrochemical sensor system.

Highly bioactive bone cement microspheres based on α-tricalcium phosphate microparticles/mesoporous bioactive glass nanoparticles: Formulation, physico-chemical characterization and in vivo bone regeneration.

Calcium phosphate cement (CPC) is a self-setting, biocompatible and osteoconductive bone cement, however its use as a bone substitute is still limited owing to its low bioactivity (i.e. its slow in vivo resorption and slow new bone formation rate) which is a challenging issue to be addressed. Herein, we report for the first time highly bioactive bone cement microspheres formulated from a cement paste containing α-tricalcium phosphate microparticles (α-TCP) and mesoporous calcium silicate bioactive glass nanoparticles (mesoporous BGn) using a water-in-oil emulsion method. Indeed, bioactive microspheres possess high potential as bone defect fillers for bone regeneration. The α-TCP microparticles were prepared by a solid state synthesis at 1400 ºC while mesoporous BGn were synthesized by template-assissted ultrasound-mediated sol-gel method. The particle size distribution of as-prepared cement microspheres was in the range of 200 - 450 µm with a sphericity index in the range of 0.92 - 0.94. The surface morphology of α-TCP microspheres revealed α-TCP micoparticles with smooth surfaces whereas α-TCP/BGn microspheres unveiled nano-roughened α-TCP microparticles. The as-prepared α-TCP/BGn cement microspheres exhibited larger specific surface area ca 18.6 m2/g, sustained release of soluble silicate (SiO44-) ions (118 ppm within a week) and high protein adsorption capacity (252 mg/g). Notably, the α-TCP/BGn cement microspheres showed excellent in vitro surface bioactivity via formation of massive amounts of bone-like hydroxyapatite spherules and aggregates on their surfaces after soaking in simulated body fluid. Importantly, the in vivo implantation of as-prepared α-TCP/BGn cement microspheres in rat calvarial critical size bone defects for 6 weeks unveiled high in vivo bioactivity in terms of substantial new bone ingrowth and significant new bone formation within the bone defect as evidenced by histological analyses, X-ray radiography and micro-computed tomography evaluations.

Evaluation of Biomechanical Properties and Morphometric Structures of the Trachea in Pigs and Rabbits.

BACKGROUND/AIM: Animals differ in the biochemical composition, attachments, and mechanical properties of tracheal cartilage. This study examined the biomechanical properties and morphological structure of the trachea of pigs, and rabbits as preclinical models. MATERIALS AND METHODS: The trachea in pigs and rabbits can be divided into four regions, cranial cervical, middle cervical, thoracic inlet, and intra-thoracic parts. RESULTS: The total number of tracheal rings in pigs and rabbits was 32-35 and 34-38 rings, respectively. The pig bronchus first branches from the trachea, reaching the cranial lobe of the lungs before branching to the main bronchus, while the rabbit bronchus branched after the main bronchus. A comparison of the posterior region of the crosssectional trachea shows that the rabbit has a C-shape with cartilage connected to the tracheal muscle, and the pig has the tracheal muscle covered with cartilage. The trachea of pigs and rabbits decreased in tracheal thickness and size from the thoracic inlet toward the lungs. The stress-strain in the longitudinal and transverse tensile test was higher in rabbits than in pigs. The tensile stress of the four regions was significantly different in the transverse tensile test (p<0.001). In the bending test, more force was required to bend pig than rabbit tracheas. Microscopic and scanning electron microscopy showed no structural differences in tracheal cartilage between the two species. CONCLUSION: These results suggest that there is great variation in morphology and physical properties of the trachea in pigs and rabbits. We found porcine tracheas have similar biomechanical properties to those of humans.

Predicting UPDRS Motor Symptoms in Individuals With Parkinson's Disease From Force Plates Using Machine Learning.

Parkinson's disease (PD) is a neurodegenerative disease that affects motor abilities with increasing severity as the disease progresses. Traditional methods for diagnosing PD include a section where a trained specialist scores qualitative symptoms using the motor subscale of the Unified Parkinson's Disease Rating Scale (UPDRS-III). The aim of this feasibility study was twofold. First, to evaluate quiet standing as an additional, out-of-clinic, objective feature to predict UPDRS-III subscores related to motor symptom severity; and second, to use quiet standing to detect the presence of motor symptoms. Force plate data were collected from 42 PD patients and 43 healthy controls during quiet standing (a task involving standing still with eyes open and closed) as a feasible task in clinics. Predicting each subscore of the UPDRS-III could aid in identifying progression of PD and provide specialists additional tools to make an informed diagnosis. Random Forest feature importance indicated that features correlated with range of center of pressure (i.e., the medial-lateral and anterior-posterior sway) were most useful in the prediction of the top PD prediction subscores of postural stability (r = 0.599; p = 0.014), hand tremor of the left hand (r = 0.650; p = 0.015), and tremor at rest of the left upper extremity (r = 0.703; p = 0.016). Quiet standing can detect body bradykinesia (AUC-ROC = 0.924) and postural stability (AUC-ROC = 0.967) with high predictability. Although there are limited data, these results should be used as a feasibility study that evaluates the predictability of individual UPDRS-III subscores using quiet standing data.

Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

Combined Delivery of Two Different Bioactive Factors Incorporated in Hydroxyapatite Microcarrier for Bone Regeneration.

BACKGROUND: The delivery of growth factors using a carrier system presents a promising and innovative tool in tissue engineering and dentistry today. Two of the foremost bioactive factors, bone morphogenetic protein-2 and vascular endothelial growth factor (VEGF), are widely applied using a ceramic scaffold. The aim of this study was to determine the use of hydroxyapatite microcarrier (MC) for dual delivery of osteogenic and angiogenic factors to accelerate hard tissue regeneration during the regenerative process. METHODS: Two MCs of different sizes were fabricated by emulsification of gelatin and alpha-tricalcium phosphate (α-TCP). The experimental group was divided based on the combination of MC size and growth factors. For investigating the in vitro properties, rat mesenchymal stem cells (rMSCs) were harvested from bone marrow of the femur and tibia. For in vivo experiments, MC with/without growth factors was applied into the standardized, 5-mm diameter defects, which were made bilaterally on the parietal bone of the rat. The animals were allowed to heal for 8 weeks, and samples were harvested and analyzed by micro-computed tomography and histology. RESULTS: Improved proliferation of rat mesenchymal stem cells was observed with VEGF loaded MC. For osteogenic differentiation, dual growth factors delivered by MC showed higher osteogenic gene expression, alkaline phosphatse production and calcium deposition. The in vivo results revealed statistically significant increase in new bone formation when dual growth factors were delivered by MC. Dual growth factors administered on a calcium phosphate matrix showed significantly enhanced osteogenic potential. CONCLUSION: We propose this system has potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of early availability.

Novel bone-mimetic nanohydroxyapatite/collagen porous scaffolds biomimetically mineralized from surface silanized mesoporous nanobioglass/collagen hybrid scaffold: Physicochemical, mechanical and in vivo evaluations.

Bone-mimetic scaffolds are receiving much interest as such scaffolds exhibit excellent biocompatibility and very close mimic to bone structure and composition. Here, novel bone-mimetic nanohydroxyapatite (nHA)/collagen (Col) porous scaffolds (nHA/Col) were prepared from surface silanized mesoporous nanobioglass (NBG)/Col hybrid scaffold by biomimetic mineralization. Surface silanized mesoporous NBG was prepared by ultrasound-assisted sol-gel method and post treatment with 3-aminopropyltriethylsilane (APTS). The surface silanized mesoporous NBG was characterized by transmission electron microscopy (TEM), transmission electron microscopy-selected area electron diffraction (TEM-SAED) and X-ray photoelectron spectroscopy (XPS). The physicochemical/mechanical characterizations of the scaffolds included scanning electron microscopy (SEM) and TEM imaging of micro/nanostructure, energy dispersive X-ray (EDX) analysis of chemical composition, TEM-SAED and X-ray diffraction/Attenuated total Reflectance-Fourier Infrared spectroscopy (XRD/ATR-FTIR) analyses of amorphous-to-crystalline transformations, thermogravimetric/differential scanning calorimetric (TGA/DSC) analyses of thermal behaviour , porosity and dynamic mechanical analyses. The presence of NBG in collagen fibrillar network enabled progressive growth of HA nanocrystals and generation of a novel bone-mimetic hybrid structures while preserving the highly porous structure of collagen scaffold. The crystallinity, crystallite size and crystal morphology of the grown HA nanocrystals were controllable by regulation of the mineralization time. Furthermore, the osteogenic properties of the non-mineralized (NBG/Col) and mineralized (nHA/Col) hybrid porous scaffolds were examined in vivo using critical-sized calvarial bone defect model in rat. Histological and micro-computed tomography (Micro-CT) analyses after 6 weeks of implantation revealed that the mineralized scaffolds possess excellent in vivo osteogenic potential compared to the non-mineralized one. Collectively, by using surface silanized mesoporous NBG hybridization with collagen fibrillar network, we successfully introduced a new approach for developing novel bone-mimetic nanohydroxyapatite/collagen hybrid scaffolds that possess significant potential for bone tissue regeneration.

Correction: Dynamic mechanical and nanofibrous topological combinatory cues designed for periodontal ligament engineering.

[This corrects the article DOI: 10.1371/journal.pone.0149967.].

SIS/aligned fibre scaffold designed to meet layered oesophageal tissue complexity and properties.

With donor organs not readily available, the need for a tissue-engineered oesophagus remains high, particularly for congenital childhood conditions such as atresia. Previous attempts have not been successful, and challenges remain. Small intestine submucosa (SIS) is an acellular matrix material with good biological properties; however, as is common with these types of materials, they demonstrate poor mechanical properties. In this work, electrospinning was performed to mechanically reinforce tubular SIS with polylactic-co-glycolic acid (PLGA) nanofibres. It was hypothesised that if attachment could be achieved between the two materials, then this would (i) improve the SIS mechanical properties, (ii) facilitate smooth muscle cell alignment to support directional growth of muscle cells and (iii) allow for the delivery of bioactive molecules (VEGF in this instance). Through a relatively simple multistage process, adhesion between the layers was achieved without chemically altering the SIS. It was also found that altering mandrel rotation speed affected the alignment of the PLGA nanofibres. SIS-PLGA scaffolds performed mechanically better than SIS alone; yield stress improvement was 200% and 400% along the longitudinal and circumferential directions, respectively. Smooth muscle cells cultured on the aligned fibres showed resultant unidirectional alignment. In vivo the SIS-PLGA scaffolds demonstrated limited foreign body reaction judged by the type and proportion of immune cells present and lack of fibrous encapsulation. The scaffolds remained intact at 4 weeks in vivo, and good cellular infiltration was observed. The incorporation of VEGF within SIS-PLGA scaffolds increased the blood vessel density of the surrounding tissues, highlighting the possible stimulation of endothelialisation by angiogenic factor delivery. Overall, the designed SIS-PLGA-VEGF hybrid scaffolds might be used as a potential matrix platform for oesophageal tissue engineering. In addition to this, achieving improved attachment between layers of acellular matrix materials and electrospun fibre layers offers the potential utility in other applications. STATEMENT OF SIGNIFICANCE: Because of its multi-layered nature and complex structure, the oesophagus tissue poses several challenges for successful clinical grafting. Therefore, it is promising to utilise tissue engineering strategies to mimic and form structural compartments for its recovery. In this context, we investigated the use of tubular small intestine submucosa (SIS) reinforced with polylactic-co-glycolic acid (PLGA) nanofibres by using electrospinning and also, amongst other parameters, the integrity of the bilayered structure created. This was carried out to facilitate smooth muscle cell alignment, support directional growth of muscle cells and allow the delivery of bioactive molecules (VEGF in this study). We evaluated this approach by using in vitro and in vivo models to determine the efficacy of this new system.

Reformulated mineral trioxide aggregate components and the assessments for use as future dental regenerative cements.

Mineral trioxide aggregate, which comprises three major inorganic components, namely, tricalcium silicate (C3S), dicalcium silicate (C2S), and tricalcium aluminate (C3A), is promising regenerative cement for dentistry. While mineral trioxide aggregate has been successfully applied in retrograde filling, the exact role of each component in the mineral trioxide aggregate system is largely unexplored. In this study, we individually synthesized the three components, namely, C3S, C2A, and C3A, and then mixed them to achieve various compositions (a total of 14 compositions including those similar to mineral trioxide aggregate). All powders were fabricated to obtain high purity. The setting reaction of all cement compositions was within 40 min, which is shorter than for commercial mineral trioxide aggregate (~150 min). Over time, the pH of the composed cements initially showed an abrupt increase and then plateaued (pH 10-12), which is a typical behavior of mineral trioxide aggregate. The compression and tensile strength of the composed cements increased (2-4 times the initial values) with time for up to 21 days in an aqueous medium, the degree to which largely depended on the composition. The cell viability test with rat mesenchymal stem cells revealed no toxicity for any composition except C3A, which contained aluminum. To confirm the in vivo biological response, cement was retro-filled into an extracted rat tooth and the complex was re-implanted. Four weeks post-operation, histological assessments revealed that C3A caused significant tissue toxicity, while good tissue compatibility was observed with the other compositions. Taken together, these results reveal that of the three major constituents of mineral trioxide aggregate, C3A generated significant toxicity in vitro and in vivo, although it accelerated setting time. This study highlights the need for careful consideration with regard to the composition of mineral trioxide aggregate, and if possible (when other properties are satisfactory), the C3A component should be avoided, which can be achieved by the mixture of individual components.

Vertical Bone Augmentation Using Three-dimensionally Printed Cap in the Rat Calvarial Partial Defect.

BACKGROUND/AIM: Lost alveolar bone is commonly restored by distraction osteogenesis or bone blocks for substantial vertical bone augmentation (VBA), that is applied in conjunction with a barrier system. This study was performed to determine whether volume control of a three-dimensional (3D) printed nylon cap in the rat calvarial partial thickness bone defect would induce qualitative and quantitative differences in vertical bone regeneration. MATERIALS AND METHODS: A rat calvarial partial thickness bone defect was prepared and the 3D cap covered the defect to induce VBA, while the control group was left without cap placement. After six weeks the animals were sacrificed, and the calvaria were prepared for micro-CT (µCT) and histology. RESULTS: Quantitative µCT results showed that our cap system has significant osteoconductive properties, and the histology slide revealed new bone filled inside the cap. CONCLUSION: The results clearly showed that this system was successful for VBA in a research animal model.

Dual growth factor-immobilized asymmetrically porous membrane for bone-to-tendon interface regeneration on rat patellar tendon avulsion model.

Insufficient repair of the bone-to-tendon interface (BTI) with structural/compositional gradients has been a significant challenge in orthopedics. In this study, dual growth factor (platelet-derived growth factor-BB [PDGF-BB] and bone morphogenetic protein-2 [BMP-2])-immobilized polycaprolactone (PCL)/Pluronic F127 asymmetrically porous membrane was fabricated to estimate its feasibility as a potential strategy for effective regeneration of BTI injury. The growth factors immobilized (via heparin-intermediated interactions) on the membrane were continuously released for up to ∼80% of the initial loading amount after 5 weeks without a significant initial burst. From the in vivo animal study using a rat patellar tendon avulsion model, it was observed that the PDGF-BB/BMP-2-immobilized membrane accelerates the regeneration of the BTI injury, probably because of the continuous release of both growth factors (biological stimuli) and their complementary effect to create a multiphasic structure (bone, fibrocartilage, and tendon) like a native structure, as well as the role of the asymmetrically porous membrane as a physical barrier (nanopore side; prevention of fibrous tissue invasion into the defect site) and scaffold (micropore side; guidance for tissue regeneration). © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 115-125, 2018.

Effect of Hydrogen Dioxide Treatment on the Osteogenic Potential of Duck-beak Bone-derived Natural Bioceramic Microparticles.

BACKGROUND/AIM: As an alternative material to the autogenous bone, duck-beak bone particle for bone substitute have been attracting great attention due to their biological properties. To deliver the most favorable outcome of medical treatment, it is essential to study the effect of various processing methods of the duck-beak bone. In this study, we compared the two deproteinizing agents for manufacturing duck-beak bone. Group 1 was treated by a conventional chemical agent (ethylenediamine) and Group 2 by hydrogen dioxide (H2O2). In vitro and in vivo experiments were conducted in parallel to compare the cytocompatibility and osteogenic capability between two processing methods. For in vitro tests, human adipose-derived mesenchymal stem cells (hAD-MSCs) were planted onto each sample and their attachment and growing were evaluated. For in vivo biocompatibility and osteogenic properties, the samples were applied on the critical-sized calvarial bone defect of rats. Group 2 showed significantly higher cell attachment but Group1 showed slightly higher cell proliferation. In in vivo tests, all groups have shown biocompatibility and increased level of osteogenic potential. However, Group 2 had significantly higher bone regeneration (p<0.05). This experiment confirmed that H2O2 can be an optimal processing method for duck-beak bone particle.

Optical imaging and anticancer chemotherapy through carbon dot created hollow mesoporous silica nanoparticles.

Multifunctional nanocarrier-based theranostics is currently considered to solve some key unmet challenges in cancer treatment. Here we report a nanocarrier platform, named carbon dot (CD) created mesoporous hollow organosilica (C-hMOS) nanoparticles, to deliver anticancer drug and to enable optical imaging. The hollow structure was formed by the removal of a nanorod core template, and at the same time, the fluorescent signal was endowed from the heat-treated organosilica network. Thanks to the hollow and mesoporous structure, the C-hMOS effectively loaded doxorubicin (DOX) for cancer chemotherapy. The DOX was released from C-hMOS highly sustainably (over 12days) and pH-dependently (pH 5.0 >pH 7.4). The DOX-loading C-hMOS internalized cancer cells efficiently (>90%), and induced cellular apoptosis including the expression of caspase-3. The treatment of C-hMOS to cancer cells enabled multi-color visualization in vitro, suggesting the possibility of cell tracing. Moreover, when injected intratumorally in mice, the C-hMOS exhibited strong optical signals in vivo along with a high optical stability (over a week). The injected C-hMOS were distributed only a fraction in liver but not in heart, lung, spleen or kidney and displayed good biocompatibility. The DOX-delivering C-hMOS significantly suppressed the in vivo tumor growth associated with apoptotic functions. Taken together, the developed C-hMOS nanoparticles can be a promising nanoplatform for drug delivery and in vivo imaging in cancer treatment. STATEMENT OF SIGNIFICANCE: Multifunctional nanoparticles that combine chemotherapeutic ability with imaging modality comprise promising platform for cancer theranostics. Here we developed a novel theranostic nanoparticle, i.e., carbon-dot created mesoporous hollow silica nanoparticle, to offer unique merit for this purpose. The in vitro and in vivo findings to support this include: i) carbon dots with 1-2nm size in situ generated discretely and uniformly within silica network, ii) hollow and mesoporous structure effective for loading of DOX at high content, iii) release behavior of DOX in a sustainable and pH-dependent manner, iv) chemotherapeutic efficacy in killing cancer cells and suppressing tumor growth through DOX delivery, and v) carbon dot induced multi-color fluorescence imaging within cells and tumor tissues. These collective multifaceted properties may facilitate the novel carbon dot nanocarriers to be a potential candidate for delivering anticancer drug and non-invasive imaging in cancer treatment.

Evaluation of the periodontal regenerative properties of patterned human periodontal ligament stem cell sheets.

PURPOSE: The aim of this study was to determine the effects of patterned human periodontal ligament stem cell (hPDLSC) sheets fabricated using a thermoresponsive substratum. METHODS: In this study, we fabricated patterned hPDLSC sheets using nanotopographical cues to modulate the alignment of the cell sheet. RESULTS: The hPDLSCs showed rapid monolayer formation on various surface pattern widths. Compared to cell sheets grown on flat surfaces, there were no significant differences in cell attachment and growth on the nanopatterned substratum. However, the patterned hPDLSC sheets showed higher periodontal ligamentogenesis-related gene expression in early stages than the unpatterned cell sheets. CONCLUSIONS: This experiment confirmed that patterned cell sheets provide flexibility in designing hPDLSC sheets, and that these stem cell sheets may be candidates for application in periodontal regenerative therapy.

Promoting angiogenesis with mesoporous microcarriers through a synergistic action of delivered silicon ion and VEGF.

Angiogenic capacity of biomaterials is a key asset to drive vascular ingrowth during tissue repair and regeneration. Here we design a unique angiogenic microcarrier based on sol-gel derived mesoporous silica. The microspheres offer a potential angiogenic stimulator, Si ion, 'intrinsically' within the chemical structure. Furthermore, the highly mesoporous nature allows the loading and release of angiogenic growth factor 'extrinsically'. The Si ion is released from the microcarriers at therapeutic ranges (over a few ppm per day), which indeed up-regulates the expression of hypoxia inducing factor 1α (HIF1α) and stabilizes it by blocking HIF-prolyl hydroxylase 2 (PHD2) in HUVECs. This in turn activates the expression of a series of proangiogenic molecules, including bFGF, VEGF, and eNOS. VEGF is incorporated effectively within the mesopores of microcarriers and is then released continuously over a couple of weeks. The Si ion and VEGF released from the microcarriers synergistically stimulate endothelial cell functions, such as cell migration, chemotactic homing, and tubular networking. Furthermore, in vivo neo-blood vessel sprouting in chicken chorioallantoic membrane model is significantly promoted by the Si/VEGF releasing microcarriers. The current study demonstrates the synergized effects of Si ion and angiogenic growth factor through a biocompatible mesoporous microsphere delivery platform, and the concept provided here may open the door to a new co-delivery system of utilizing ions with growth factors for tissue repair and regeneration.

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers.

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS.

Angiogenic Effects of Collagen/Mesoporous Nanoparticle Composite Scaffold Delivering VEGF165.

Vascularization is a key issue for the success of tissue engineering to repair damaged tissue. In this study, we report a composite scaffold delivering angiogenic factor for this purpose. Vascular endothelial growth factor (VEGF) was loaded on mesoporous silica nanoparticle (MSN), which was then incorporated within a type I collagen sponge, to produce collagen/MSN/VEGF (CMV) scaffold. The CMV composite scaffold could release VEGF sustainably over the test period of 28 days. The release of VEGF improved the cell proliferation. Moreover, the in vivo angiogenesis of the scaffold, as studied by the chick chorioallantoic membrane (CAM) model, showed that the VEGF-releasing scaffold induced significantly increased number of blood vessel complexes when compared with VEGF-free scaffold. The composite scaffold showed good biocompatibility, as examined in rat subcutaneous tissue. These results demonstrate that the CMV scaffold with VEGF-releasing capacity can be potentially used to stimulate angiogenesis and tissue repair.

Surface guidance of stem cell behavior: Chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitro and bone formation in vivo.

UNLABELLED: Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor α5ß1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs. STATEMENT OF SIGNIFICANCE: Organic coatings have been proposed as a solution to foster osseointegration of orthopedic implants. Among them, extracellular matrix-derived peptide motifs are an interesting biomimetic strategy to harness cell-surface interactions. Nonetheless, the combination of multiple peptide motifs in a controlled manner is essential to achieve receptor specificity and fully exploit the potentiality of synthetic peptides. Herein, we covalently graft to titanium a double branched molecule to guide stem cell fate in vitro and generate an osseoinductive titanium surface in vivo. Such synthetic ligand allows for the simultaneous presentation of two bioactive motifs, thus is ideal to test the effect of synergic sequences, such as RGD and PHSRN, and is a clear example of the versatility and feasibility of rationally designed biomolecules.

Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration.

The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ∼73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.