Maternal administration of erythromycin fails to eradicate intrauterine ureaplasma infection in an ovine model

Erythromycin is the standard antibiotic used for treatment of Ureaplasma species during 3 pregnancy; however, maternally administered erythromycin may be ineffective at eliminating 4 intra-amniotic ureaplasma infections. We asked if erythromycin would eradicate intra-amniotic 5 ureaplasma infections in pregnant sheep. At 50 days of gestation (d, term=150d) pregnant ewes 6 received intra-amniotic injections of erythromycin-sensitive U. parvum serovar 3 (n=16) or 10B 7 medium (n=16). At 100d, amniocentesis was performed; five fetal losses (ureaplasma group: 8 n=4; 10B group: n=1) had occurred by this time. Remaining ewes were allocated into treatment 9 subgroups: medium only (M, n=7); medium and erythromycin (M/E, n=8); ureaplasma only (Up, 10 n=6) or ureaplasma and erythromycin (Up/E, n=6). Erythromycin was administered intra11 muscularly (500 mg), eight-hourly for four days (100d-104d). Amniotic fluid samples were 12 collected at 105d. At 125d preterm fetuses were surgically delivered and specimens were 13 collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid 14 chromatography-mass spectrometry. Ureaplasmas were isolated from the amniotic fluid, 15 chorioamnion and fetal lung of animals from the Up and Up/E groups, however, the numbers of 16 U. parvum recovered were not different between these groups. Inflammation in the 17 chorioamnion, cord and fetal lung was increased in ureaplasma-exposed animals compared to 18 controls, but was not different between the Up and Up/E groups. Erythromycin was detected in 19 amniotic fluid samples, although concentrations were low (<10-76 ng/mL). This study 20 demonstrates that maternally administered erythromycin does not eradicate chronic, intra- amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially due to 22 the poor placental passage of erythromycin.

In situ preparation and protein delivery of silicate/alginate composite microspheres with core-shell structure

It is predicted that with increased life expectancy in the developed world, there will be a greater demand for synthetic materials to repair or regenerate lost, injured or diseased bone (Hench & Thompson 2010). There are still few synthetic materials having true bone inductivity, which limits their application for bone regeneration, especially in large-size bone defects. To solve this problem, growth factors, such as bone morphogenetic proteins (BMPs), have been incorporated into synthetic materials in order to stimulate de novo bone formation in the center of large-size bone defects. The greatest obstacle with this approach is that the rapid diffusion of the protein from the carrier material, leading to a precipitous loss of bioactivity; the result is often insufficient local induction or failure of bone regeneration (Wei et al. 2007). It is critical that the protein is loaded in the carrier material in conditions which maintains its bioactivity (van de Manakker et al. 2009). For this reason, the efficient loading and controlled release of a protein from a synthetic material has remained a significant challenge. The use of microspheres as protein/drug carriers has received considerable attention in recent years (Lee et al. 2010; Pareta & Edirisinghe 2006; Wu & Zreiqat 2010). Compared to macroporous block scaffolds, the chief advantage of microspheres is their superior protein-delivery properties and ability to fill bone defects with irregular and complex shapes and sizes. Upon implantation, the microspheres are easily conformed to the irregular implant site, and the interstices between the particles provide space for both tissue and vascular ingrowth, which are important for effective and functional bone regeneration (Hsu et al. 1999). Alginates are natural polysaccharides and their production does not have the implicit risk of contamination with allo or xeno-proteins or viruses (Xie et al. 2010). Because alginate is generally cytocompatible, it has been used extensively in medicine, including cell therapy and tissue engineering applications (Tampieri et al. 2005; Xie et al. 2010; Xu et al. 2007). Calcium cross-linked alginate hydrogel is considered a promising material as a delivery matrix for drugs and proteins, since its gel microspheres form readily in aqueous solutions at room temperature, eliminating the need for harsh organic solvents, thereby maintaining the bioactivity of proteins in the process of loading into the microspheres (Jay & Saltzman 2009; Kikuchi et al. 1999). In addition, calcium cross-linked alginate hydrogel is degradable under physiological conditions (Kibat PG et al. 1990; Park K et al. 1993), which makes alginate stand out as an attractive candidate material for the protein carrier and bone regeneration (Hosoya et al. 2004; Matsuno et al. 2008; Turco et al. 2009). However, the major disadvantages of alginate microspheres is their low loading efficiency and also rapid release of proteins due to the mesh-like networks of the gel (Halder et al. 2005). Previous studies have shown that a core-shell structure in drug/protein carriers can overcome the issues of limited loading efficiencies and rapid release of drug or protein (Chang et al. 2010; Molvinger et al. 2004; Soppimath et al. 2007). We therefore hypothesized that introducing a core-shell structure into the alginate microspheres could solve the shortcomings of the pure alginate. Calcium silicate (CS) has been tested as a biodegradable biomaterial for bone tissue regeneration. CS is capable of inducing bone-like apatite formation in simulated body fluid (SBF) and its apatite-formation rate in SBF is faster than that of Bioglass® and A-W glass-ceramics (De Aza et al. 2000; Siriphannon et al. 2002). Titanium alloys plasma-spray coated with CS have excellent in vivo bioactivity (Xue et al. 2005) and porous CS scaffolds have enhanced in vivo bone formation ability compared to porous β-tricalcium phosphate ceramics (Xu et al. 2008). In light of the many advantages of this material, we decided to prepare CS/alginate composite microspheres by combining a CS shell with an alginate core to improve their protein delivery and mineralization for potential protein delivery and bone repair applications

Porous zirconia scaffold modified with mesoporous bioglass coating

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute as its high mechanical strength. However, porous YSZ bodies are biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance the bioactivity. In this study, the porous zirconia scaffolds were prepared by infiltration of Acrylonitrile Butadiene Styrene (ABS) scaffolds with 3 mol% yttria stabilized zirconia slurry. After sintering, a method of sol-gel dip coating was involved to make coating layer of mesoporous bioglass (MBGs). The porous zirconia without the coating had high porosities of 60.1% to 63.8%, and most macropores were interconnected with pore sizes of 0.5-0.8mm. The porous zirconia had compressive strengths of 9.07-9.90MPa. Moreover, the average coating thickness was about 7μm. There is no significant change of compressive strength for the porous zirconia with mesoporous biogalss coating. The bone marrow stromal cell (BMSC) proliferation test showed both uncoated and coated zirconia scaffolds have good biocompatibility. The scanning electron microscope (SEM) micrographs and the compositional analysis graphs demonstrated that after testing in the simulated body fluid (SBF) for 7 days, the apatite formation occurred on the coating surface. Thus, porous zirconia-based ceramics were modified with bioactive coating of mesoporous bioglass for potential biomedical applications.

Nanostructured mesoporous bioglass coated zirconia scaffolds for bone tissue engineering

For the filling and reconstruction of non-healing bone defects, the application of porous ceramic scaffold as bone substitutes is considered to be a reasonable choice. In bone tissue engineering, an ideal scaffold must satisfy several criterias such as open porosity, having high compressive strength (it depends where in body, and if external fixatures are used) and the practicability for cell migration. Many researchers have focused on enhancing the mechanical properties of hydroxyapatite scaffolds by combining it with other biomaterials, such as bioglass and polymers. Nevertheless, there is still a lack of suitable scaffolds based on porous biomaterials. In this study, zirconia scaffolds from two different templates (polyurethane (PU) and Acrylonitrile Butadiene Styrene (ABS) templates) were successfully fabricated with dissimilar fabrication techniques. The scaffold surfaces were further modified with mesoporous bioglass for the purpose of bone tissue engineering. In the study of PU template scaffold, high porosity (~88%) sol-gel derived yttria-stabilized zirconia (YSZ) scaffold was prepared by a polyurethane (PU) foam replica method using sol-gel derived zirconia for the first time, and double coated with Mesoporous Bioglass (MBGs) coating. For the ABS template scaffold, two types of templates (cube and cylinder) with different strut spacings were used and fabricated by a 3D Rapid Prototyper. Subsequently, zirconia scaffolds with low porosity (63±2.8% to 68±2.5%) were fabricated by embedding the zirconia powder slurry into the ABS templates and burning out the ABS to produce a uniform porous structure. The zirconia scaffolds were double coated with mesoporous bioglass by dip coating for the first time. The porosities of the scaffolds were calculated before and after coating. The microstructures were then examined using scanning electron microscopy and the mechanical properties were evaluated using compressive test. Accordingly, relationships between microstructure, processing and mechanical behaviour of the porous zirconia was discussed. Scaffold biocompatibility and bioactivity was also evaluated using a bone marrow stromal cell (BMSC) proliferation test and a simulated body fluid test.

The role of the multiple banded antigen of ureaplasma parvum in intra-amniotic infection : major virulence factor or decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Biochemical profiling of proteins and metabolites in wound exudate from chronic wound environments

The lack of fundamental knowledge on the biological processes associated with wound healing represents a significant challenge. Understanding the biochemical changes that occur within a chronic wound could provide insights into the wound environment and enable more effective wound management. We report on the stability of wound fluid samples under various conditions and describe a high-throughput approach to investigate the altered biochemical state within wound samples collected from various types of chronic, ulcerated wounds. Furthermore, we discuss the viability of this approach in the early stages of wound sample protein and metabolite profiling and subsequent biomarker discovery. This approach will facilitate the detection of factors that may correlate with wound severity and/or could be used to monitor the response to a particular treatment.

Preparation of mesoporous bioglass coated zirconia scaffold for bone tissue engineering

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute due to its high mechanical strength. However, porous YSZ is biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance its bioactivity. In this study, porous YSZ scaffolds were prepared using a replication technique and then coated with mesoporous bioglass due to its excellent bioactivity. The microstructures were examined using scanning electron microscopy and the mechanical strength was evaluated via compression test. The biocompatibility and bioactivity were also evaluated using bone marrow stromal cell (BMSC) proliferation test and simulated body fluid test.

Investigation on bioactivity of titanium dioxide thin films fabricated by electron cyclotron resonance plasma aided pulsed laser deposition

Titanium dioxide thin films with a rutile crystallinite size around 20 nm were fabricated by pulsed laser deposition (PLD) aided with an electron cyclotron resonance (ECR) plasma. With annealing treatment, the crystal size of the rutile crystallinite increased to 100 nm. The apatite-forming ability of the films as deposited and after annealing was investigated in a kind of simulated body fluid with ion concentrations nearly equal to those of human blood plasma. The results indicate that ECR aided PLD is an effective way both to fabricate bioactive titanium dioxide thin films and to optimize the bioactivity of titanium dioxide, with both crystal size and defects of the film taken into account.

Reactive plasma-aided RF sputtering deposition of hydroxyapatite bio-implant coatings

The plasma-assisted RF sputtering deposition of a biocompatible, functionally graded calcium phosphate bioceramic on a Ti6A14 V orthopedic alloy is reported. The chemical composition and presence of hydroxyapatite (HA), CaTiO3, and CaO mineral phases can be effectively controlled by the process parameters. At higher DC biases, the ratio [Ca]/[P] and the amount of CaO increase, whereas the HA content decreases. Optical emission spectroscopy suggests that CaO+ is the dominant species that responds to negative DC bias and controls calcium content. Biocompatibility tests in simulated body fluid confirm a positive biomimetic response evidenced by in-growth of an apatite layer after 24 h of immersion.

RF plasma sputtering deposition of hydroxyapatite bioceramics : synthesis, performance, and biocompatibility

Hydroxyapatite (HA) coatings have numerous applications in orthopedics and dentistry, owing to their excellent ability to promote stronger implant fixation and faster bone tissue ingrowth and remodeling. Thermal plasma spray and other plasma-assisted techniques have recently been used to synthesize various calcium phosphate-based bioceramics. Despite notable recent achievements in the desired stoichiometry, phase composition, mechanical, structural, and bio-compatible properties, it is rather difficult to combine all of the above features in a single coating. For example, many existing plasma-sprayed HA coatings fall short in meeting the requirements of grain size and crystallinity, and as such are subject to enhanced resorption in body fluid. On the other hand, relatively poor interfacial bonding and stability is an obstacle to the application of the HA coatings in high load bearing Ti6Al4V knee joint implants. Here, we report on an alternative: a plasma-assisted, concurrent, sputtering deposition technique for high performance biocompatible HA coatings on Ti6Al4V implant alloy. The plasma-assisted RF magnetron co-sputtering deposition method allows one to simultaneously achieve most of the desired attributes of the biomimetic material and overcome the aforementioned problems. This article details the film synthesis process specifications, extensive analytical characterization of the material's properties, mechanical testing, simulated body fluid assessments, biocompatibility and cytocompatibility of the HA-coated Ti6Al4V orthopedic alloy. The means of optimization of the plasma and deposition process parameters to achieve the desired attributes and performance of the HA coating, as well as future challenges in clinical applications are also discussed.

Development and characterization of a novel, antimicrobial, sterile hydrogel dressing for burn wounds : single-step production with gamma irradiation creates silver nanoparticles and radical polymerization

Patients with burn wounds are susceptible to wound infection and sepsis. This research introduces a novel burn wound dressing that contains silver nanoparticles (SNPs) to treat infection in a 2-acrylamido-2-methylpropane sulfonic acid sodium salt (AMPS-Na(+) ) hydrogel. Silver nitrate was dissolved in AMPS-Na(+) solution and then exposed to gamma irradiation to form SNP-infused hydrogels. The gamma irradiation results in a cross-linked polymeric network of sterile hydrogel dressing and a reduction of silver ions to form SNPs infused in the hydrogel in a one-step process. About 80% of the total silver was released from the hydrogels after 72 h immersion in simulated body fluid solution; therefore, they could be used on wounds for up to 3 days. All the hydrogels were found to be nontoxic to normal human dermal fibroblast cells. The silver-loaded hydrogels had good inhibitory action against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Results from a pilot study on a porcine burn model showed that the 5-mM silver hydrogel was efficient at preventing bacterial colonization of wounds, and the results were comparable to the commercially available silver dressings (Acticoat(TM) , PolyMem Silver(®) ). These results support its use as a potential burn wound dressing.

Transfer of Dietary Plant Natural Flavor Compounds From Essential Oils to Maternal Fluids in Pigs

Fetal flavor conditioning during the perinatal stage could be essential at the time of the weaning to reduce the stress and improve the feed intake in pigs. The transfer of flavor compounds from maternal diet to amniotic fluid and milk has been shown in behavioral experiments, but not through analytical procedures such as gas chromatography–mass spectrometry (GC–MS). The aim of the experiment was to trace the principal essential oils compounds supplied in the diet in maternal fluids. Twenty Large White sows around their 104th gestational day were allocated to individual farrowing crates. Two groups of 10 sows were fed either a standard gestation diet or the same diet supplemented with a mix of 8 essential oils at a rate of 1kg/ton during the last 10 days of gestation. At approximately the 113th gestational day, animals were individually treated with 10mg of Lutalyse IM was to induce farrowing. Fresh amniotic fluid was collected during the farrowing in 100-mL glass bottles and immediately stored at −20 °C freezer. During the second lactation day, 10–20 IU of Oxytocin IM was administered to each sow to facilitate collection of milk samples in 20-mL glass bottles. The samples were stored at −20 °C until analyzed by GC–MS. The presence of significant amounts of principal components of all the essential oils except one were found in the milk and amniotic fluid samples of the treated sows relative to the control sows. Our data prove the transfer of selected dietary flavors to maternal fluids and sets the scenario for further trials to manipulate postweaning behavior in piglets.

Additively manufactured device for dynamic culture of large arrays of 3D tissue engineered constructs

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.

Topical tacrolimus in atopic dermatitis : Effects of long-term treatment on skin and respiratory symptoms

Objective: Patients with atopic dermatitis often have a poor long-term response to conventional topical or systemic treatments. Staphylococcal superinfections, skin atrophy due to corticosteroid use, and asthma and allergic rhinitis are common. Only a few, usually short-term, studies have addressed the effects of different treatments on these problems. Tacrolimus ointment is the first topical compound suitable for long-term treatment. The aim of this thesis was to evaluate the effects of long-term topical tacrolimus treatment on cutaneous staphylococcal colonization, collagen synthesis, and symptoms and signs of asthma and allergic rhinitis. Methods: Patients with moderate-to-severe atopic dermatitis were treated with intermittent 0.1% tacrolimus ointment in prospective, open studies lasting for 6 to 48 months. In Study I, cutaneous staphylococcal colonization was followed for 6 to 12 months. In Study II, skin thickness and collagen synthesis were followed by skin ultrasound and procollagen I and III propeptide concentrations of suction blister fluid samples for 12 to 24 months and compared with a group of corticosteroid-treated atopic dermatitis patients and with a group of healthy subjects. Study III was a cross-sectional study of the occurrence of respiratory symptoms, bronchial hyper-responsiveness, and sputum eosinophilia in atopic dermatitis patients and healthy controls. In Study V, the same parameters as in Study III were assessed in atopic dermatitis patients before and after 12 to 48 months of topical tacrolimus treatment. Study IV was a retrospective follow-up of the effect of tacrolimus 0.03% ointment on severe atopic blepharoconjunctivitis and conjunctival cytology. Results: The clinical response to topical tacrolimus was very good in all studies (p≤0.008). Staphylococcal colonization decreased significantly, and the effect was sustained throughout the study (p=0.01). Skin thickness (p<0.001) and markers of collagen synthesis (p<0.001) increased in the tacrolimus-treated patients significantly, whereas they decreased or remained unchanged in the corticosteroid-treated controls. Symptoms of asthma and allergic rhinitis (p<0.0001), bronchial hyper-responsiveness (p<0.0001), and sputum eosinophilia (p<0.0001) were significantly more common in patients with atopic dermatitis than in healthy controls, especially in subjects with positive skin prick tests or elevated serum immunoglobulin E. During topical tacrolimus treatment the asthma and rhinitis (p=0.005 and p=0.002) symptoms and bronchial hyper-responsiveness (p=0.02) decreased significantly, and serum immunoglobulin E and sputum eosinophils showed a decreasing trend in patients with the best treatment response. Treatment of atopic blepharoconjunctivitis resulted in a marked clinical response and a significant decrease in eosinophils, lymphocytes, and neutrophils in the conjunctival cytology samples. No significant adverse effects or increase in skin infections occurred in any study. Conclusions: The studies included in this thesis, except the study showing an increase in skin collagen synthesis in tacrolimus-treated patients, were uncontrolled, warranting certain reservations. The results suggest, however, that tacrolimus ointment has several beneficial effects in the long-term intermittent treatment of atopic dermatitis. Tacrolimus ointment efficiently suppresses the T cell-induced inflammation of atopic dermatitis. It has a normalizing effect on the function of the skin measured by the decrease in staphylococcal colonization. It does not cause skin atrophy as do corticosteroids but restores the skin collagen synthesis in patients who have used corticosteroids. Tacrolimus ointment has no marked systemic effect, as the absorption of the drug is minimal and decreases along with skin improvement. The effects on the airway: decrease in bronchial hyper-responsiveness and respiratory symptoms, can be speculated to be caused by the decrease in T cell trafficking from the skin to the respiratory tissues as the skin inflammation resolves, as well as inhibition of epicutaneous invasion of various antigens causing systemic sensitization when the skin barrier is disrupted as in atopic dermatitis. Patients with moderate-to-severe atopic dermatitis seem to benefit from efficient long-term treatment with topical tacrolimus.