Polysaccharide-based (kappa carrageenan/carboxymethyl chitosan) nanofibrous membrane loaded with antifibrinolytic drug for rapid hemostasis- in vitro and in vivo evaluation.

This work aimed to establish a novel membrane consisting of hemostatic polysaccharides, kappa-carrageenan (KC), and carboxymethyl chitosan (CMC) in tandem with polyvinyl alcohol that spun together as a matrix and loaded with tranexamic acid (TXA) as antifibrinolytic agent for further coagulation effect during and after oral surgeries. The electrospinning of KC was done for the first time and in comparison of CMC has better hemostatic efficacy. The effect of the hemostat was investigated by its surface morphology (SEM), FTIR/ATR analysis, swelling behavior in both PBS and blood, hydrophilicity, porosity, mechanical properties, and cumulative release rate. The effect of materials and the drug concentration ratio were considered. The effect of acetic acid percent in aqueous solutions of CMC/PVA and KC/PVA on morphology was investigated. The cell culture assay showed that all membranes interacted well (98 %) with fibroblast cells attached and grown on the fabricated substrate. Furthermore, the membranes are evaluated by clotting time, whole blood clotting, hemocompatibility, and platelet and RBC adhesion tests. Also, the hemostatic performance of the membrane was analyzed in vivo, using the tail and liver bleeding model in rats. Therefore, TXA loading into CMC and KC dressing could be an attractive hemostatic system for various clinical applications.

Three-dimensional biomimetic reinforced chitosan/gelatin composite scaffolds containing PLA nano/microfibers for soft tissue engineering application.

In the current study, we successfully prepared chitosan/gelatin composite scaffolds reinforced by centrifugally spun polylactic acid (PLA) chopped nano/microfibers (PLA-CFs). Herein, different amounts of PLA-CFs (0 %, 1 %, 2 %, 3 %, and 4 % w/v) dispersed in chitosan/gelatin solution were used. Morphological characterization of prepared scaffolds revealed that at the initial stage of adding PLA-CFs, the chopped fibers were localized at the wall of the pores; however, as the fiber load increased, aggregations of chopped-fibers could be seen. Also, mechanical evaluation of scaffolds in terms of compression and tensile mode showed that samples reinforced with 2 % PLA-CFs had enhanced mechanical properties. Indeed, its tensile strength increased from 123.8 to 247.2 kPa for dry and 18.9 to 48.6 kPa for wet conditions. Furthermore, the tensile modulus associated with both conditions increased from 2.99 MPa and 44.5 kPa to 6.43 MPa and 158.4 kPa, respectively. The results of cell culture studies also confirmed that the prepared composite scaffold exhibited appropriate biocompatibility, cell proliferation and migration. The cell infiltration study of the samples revealed that scaffolds reinforced with 2 % PLA-CFs had significantly better cell penetration and distribution compared with the control ones on both days (7 and 14).

Polyhydroxybutyrate-starch/carbon nanotube electrospun nanocomposite: A highly potential scaffold for bone tissue engineering applications.

Blend nanofibers composed of synthetic and natural polymers with carbon nanomaterial, have a great potential for bone tissue engineering. In this study, the electrospun nanocomposite scaffolds based on polyhydroxybutyrate(PHB)-Starch-multiwalled carbon nanotubes (MWCNTs) were fabricated with different concentrations of MWCNTs including 0.5, 0.75 and 1 wt%. The synthesized scaffolds were characterized in terms of morphology, porosity, thermal and mechanical properties, biodegradation, bioactivity, and cell behavior. The effect of the developed structures on MG63 cells was determined by real-time PCR quantification of collagen type I, osteocalcin, osteopontin and osteonectin genes. Our results showed that the scaffold containing 1 wt% MWCNTs presented the lowest fiber diameter (124 ± 44 nm) with a porosity percentage above 80 % and the highest tensile strength (24.37 ± 0.22 MPa). The addition of MWCNTs has a positive effect on surface roughness and hydrophilicity. The formation of calcium phosphate sediments on the surface of the scaffolds after immersion in SBF is observed by SEM and verified by EDS and XRD analysis.MG63 cells were well cultured on the scaffold containing MWCNTs and presented more cell viability, ALP secretion, calcium deposition and gene expression compared to the scaffolds without MWCNTs. The PHB-starch-1wt.%MWCNTs scaffold can be considerable for studies of supplemental bone tissue engineering applications.

Multi-antibacterial agent-based electrospun polycaprolactone for active wound dressing.

Today, due to the greater knowledge of the side effects of chemical drugs and the favorable pharmacological properties of herbal compounds, the use of these compounds is increasing. Since wounds need fast and efficient healing, wound dressing fabrication methods play an important role in wound healing. In this research, electrospinning process was used to prepare samples. Natural antibacterial compounds, such as curcumin, piperine, eugenol, and rutin were loaded in electrospun nano-fibrous based on polycaprolactone. Three-component novel systems of curcumin-piperine-eugenol (PCPiEu), and curcumin-piperine-rutin (PCPiR) were designed and prepared. Their synergistic effect was investigated and also compared with one- and two-component systems. The results showed that medium diameter nanofibers of PCPiEu and PCPiR samples was 198.38 and 142.60, respectively, and they were obtained in smooth, uniform and bead free morphology using optimization of process parameters. The amount of water absorption and water vapor permeability of the three-component samples were in the appropriate range (8.33-10.42 mg cm2 h-1) for wound dressings. The mechanical properties of samples were reduced compared to the control sample, which required further investigation. Antibacterial tests showed good results for partial toxicity of PCPiEu and PCPiR samples. Antibacterial tests showed minor toxicity in PCPiR samples and good results were obtained for PCPiEu samples. In addition, the results showed that PCPiEu and PCPiR samples exhibited antibacterial activity against Gram-positive bacterium Staphylococcus aureus and Gram-negative Enterococcus faecalis bacterium, so that killing ability of 74% and 75% against Gram-positive bacterium and 99.47% and 96.88% against Gram-negative bacterium were obtained for these three systems, respectively.

Evaluation of the effects of starch on polyhydroxybutyrate electrospun scaffolds for bone tissue engineering applications.

Efficient design for bone tissue engineering requires an understanding of the appropriate selection of biomimetic natural or synthetic materials and scalable fabrication technologies. In this research, poly (3-hydroxybutyrate) (PHB) and starch (5-15 wt%) as biological macromolecules were used to fabricate novel biomimetic scaffolds by electrospinning method. SEM results of electrospun scaffolds revealed bead-free nanofibers and three-dimensional homogenous structures with highly interconnected pores. Results of FTIR and Raman demonstrated that there were hydrogen bonds between the two polymers. The tensile strength of scaffolds was significantly improved by adding starch up to 10 wt%, from 3.05 to 15.54 MPa. In vitro degradation and hydrophilicity of the scaffolds were improved with the presence of starch. The viability and proliferation of MG-63 cells and alkaline phosphatase (ALP) activity were remarkably increased in the PHB-starch scaffolds compared to the PHB and control samples. The mineralization and calcium deposition of MG-63 cells were confirmed by alizarin red staining. It is concluded that PHB/starch electrospun scaffold could be a good candidate for bone tissue engineering applications.

A simple, green chemistry technology for fabrication of tissue-engineered scaffolds based on mussel-inspired 3D centrifugal spun.

The fabrication of 3D fibrous scaffolds with highly interconnected pores has been crucial in the development of tissue regeneration techniques. The present study describes the fabrication of 3D fibrous scaffolds by freeze-drying of polydopamine (PDA) coated centrifugal spun gelatin fibers. We wanted to combine the mussel-inspired chemistry, Maillard reaction, and the 3D microstructural advantages of centrifugal spun fibers to develop the green fibrous scaffolds at low cost, high speed, and desired mold shape. The resultant PDA-gelatin fibers exhibited a smooth 3D microstructure with a uniform formation of PDA thin ad-layer that enhanced the mechanical properties and stability of the scaffolds, and thereby decreased the degradation rate. All scaffolds showed promising properties including good dimensional and mechanical stability under wet state, optimal porosity over 94%, and high water uptake of approximately 1500%. The results of cell culture studies, further confirmed that all scaffolds exhibited appropriate biocompatibility, cell proliferation, migration, and infiltration. Particularly, the PDA-coated scaffolds showed a significant enhancement in proliferation, migration, and infiltration of HDF-GFP+ cells. These results show that a 3D porous fibrous scaffold with simplifying tunable density and desirable shape on a large scale can be readily prepared for different fields of tissue engineering applications.

PEGylated curcumin-loaded nanofibrous mats with controlled burst release through bead knot-on-spring design.

APEGylatedcurcumin (PCU) loaded electrospuns based on poly(ε-caprolactone) (PCL) andpolyvinyl alcohol (PVA) were fabricated for wound dressing applications. The main reason for this wound dressing design is antibacterialactivity enhancement, and wound exudates management. PEGylation increases curcuminsantibacterial properties and PVA can help exudates management. For optimal wound dressing, first, response surface methodology (RSM) was applied to optimize the electrospinning parameters to achieve appropriate nanofibrous mats. Then a three-layer electrospun was designed by considering the water absorbability, PCU release profile as well as antibacterial and biocompatibility of the final wound dressing. The burst release in controlled release systems could be evaluated for prevention of the higher initial drug release and control the effective life time. The PCU release results illustrated that the bead knot plays a positive role in controlling the release profile andby increase in the number of beads per unit area from 3000 to 9000 mm-2,the PCU burst release will be reduced; Also in vitro studies show that optimized three-layer dressing based on PCL/PVA/PCU can support water vapour transmission rate in optimal range and also absorb more than three times exudates in comparison with mono-layerdressing. Antibacterial tests show that the electrospun wound dressing containing 5% PCU exhibits100% antibacterial activityas well as cell viability level within an acceptable range.

Spatial epidemiology of HIV-hepatitis co-infection in the State of Michigan: a cohort study.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is a continuing global public health threat affecting millions of individuals. In 2009, 33.3 million people worldwide were living with human immunodeficiency virus (HIV) infection. HIV-infected individuals are at an increased risk of acquiring hepatitis B and hepatitis C viral (HBV, HCV)infections because of shared transmission routes. The purpose of this study was to identify geographical clusters of HIV-(HBV/HCV) co-infection in the State of Michigan. METHODS: Retrospective cohort data on HIV-infected individuals were matched to all hepatitis B and C cases in Michigan during the period of January 1, 2006 through December 31, 2009. A prevalence map of HIV infection was created and spatial clusters of HIV-hepatitis B or C co-infection were detected using GeoDa's bivariate local Moran's I and SaTScan's discrete Poisson model. RESULTS: A bivariate cluster of high prevalence HIV and hepatitis B or C was identified in the Detroit Metropolitan Area and surrounding counties. A Poisson cluster of HIV-hepatitis B or C co-infection was identified, relative risk (RR) = 1.38 (p = 0.029) in the western and northwestern counties of Lower Michigan, controlling for sex, race, and AIDS status. CONCLUSION: This study identified elevated HIV-hepatitis B or C co-infection unexplained by sex, race or AIDS status in counties outside of the Detroit Metropolitan Area where HIV prevalence was highest in Michigan. The findings from this study may be used to target future public health policy and healthcare interventions for HIV-hepatitis co-infection in these areas.

Rationalization of specific structure formation in electrospinning process: Study on nano-fibrous PCL- and PLGA-based scaffolds.

Formation of specific structures on poly-ɛ-caprolactone (PCL) and poly lactide-co-glycolide (PLGA) based electrospun mats is rationalized and the effect of interactive parameters; high voltage and flow rate on unique surface topography is evaluated. By increasing the collecting time in electrospinning process and enhancing fiber to fiber repulsion, surface characteristics of mats changes from nano- to micro-topography. In this study surface topography of the fabricated mats based on PCL and PLGA were assessed using AFM and SEM techniques to display the distinct phenomenon occurs on collected random fibers. In this research the rationale behind the formation of bump and flower like structures on fibrous mats was discussed. Because of great potential application of the fabricated substrates in the fields of medical purposes, cell-matrix interaction was evaluated and in vitro biological test with human dermal fibroblast and mouse L929 fibroblast cells was performed to study the cell responses to different roughness of nano-fibers collected at different time intervals. Our results show that after 7 days, cell proliferation is improved on PCL collected at 40 min in the case of human fibroblast cells and on PCL collected in 70 min in the case of L929 mouse fibroblast cells.

Comparing supportive properties of poly lactic-co-glycolic acid (PLGA), PLGA/collagen and human amniotic membrane for human urothelial and smooth muscle cells engineering.

PURPOSE: To compare human urothelial and smooth muscle cells attachment and proliferation using three different matrices; poly lactic-co-glycolic acid (PLGA), PLGA/collagen and human amniotic membrane (hAM). MATERIALS AND METHODS: Human urothelial and smooth muscle cells were cultured and examined for expression of urothelium (pancytokeratin and uroplakin III) and smooth muscle cells [desmin and alpha smooth muscle actin (α-SMA)] markers. Cells were cultured on three scaffolds; PLGA, PLGA/collagen and hAM. Thereafter, they were analyzed for cell growth on days 1, 3, 7, 14 and 21 after seeding by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Scaffolds were fixed and processed for hematoxylin and eosin (H&E) staining and immunohistochemistry against their cell specific markers after 7 and 14 days of culture. RESULTS: MTT assay results revealed that collagen has improved cell attachment features of PLGA and led to significant increase of MTT signal in PLGA/collagen compared to PLGA (P < .001) and hAM (P < .001). hAM was a weaker matrix for both cell types as demonstrated in MTT assay and scanning electron microscope (SEM) images. SEM micrographs showed normal phenotype and distribution on PLGA and PLGA/collagen. In the same line, cells formed a well-developed layer either on PLGA or PLGA/collagen, which maintained expression of their corresponding markers. CONCLUSION: Our findings demonstrated significant improvement of cell attachment and growth achieved by collagen coating (PLGA/collagen) compared to PLGA and hAM. hAM despite of its natural entity was a weaker matrix for bladder engineering purposes.

The effect of topography on differentiation fates of matrigel-coated mouse embryonic stem cells cultured on PLGA nanofibrous scaffolds.

Due to pluripotency of embryonic stem (ES) cells, these cells are an invaluable in vitro model that investigates the influence of different physical and chemical cues on differentiation/development pathway of specialized cells. We sought the effect of roughness and alignment, as topomorpholocial properties of scaffolds on differentiation of green fluorescent protein-expressing ES (GFP-ES) cells into three germ layers derivates simultaneously. Furthermore, the effect of Matrigel as a natural extracellular matrix in combination with poly(lactic-co-glycolic acid) (PLGA) nanofibrous scaffolds on differentiation of mouse ES cells has been investigated. The PLGA nanofibrous scaffolds with different height and distribution of roughness and alignments were fabricated. Then, the different cell differentiation fats of GFP-ES cells plated on PLGA and PLGA/Matrigel scaffolds were analyzed by gene expression profiling. The findings demonstrated that distinct ranges of roughness, height, and distribution can support/promote a specific cell differentiation fate on scaffolds. Coating of scaffolds with Matrigel has a synergistic effect in differentiation of mesoderm-derived cells and germ cells from ES cells, whereas it inhibits the derivation of endodermal cell lineages. It was concluded that the topomorpholocial cues such as roughness and alignment should be considered in addition to other scaffolds properties to design an efficient electrospun scaffold for specific tissue engineering.

Case-control study of disease determinants for non-typhoidal Salmonella infections among Michigan children.

BACKGROUND: Infections with Salmonella serotypes continue to be a significant global public health problem. In addition to contaminated foods, several other sources contribute to infections with Salmonella serotypes. We have assessed the role of socioeconomic factors, exposure to food, and environmental sources in the etiology of non-typhoidal Salmonella infections in Michigan children. FINDINGS: A case-control study among Michigan children aged

Cloning of Salmonella enterica serovar Enteritidis fimbrial protein SefA as a surface protein in Escherichia coli confers the ability to attach to eukaryotic cell lines.

The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

The role of neighborhood level socioeconomic characteristics in Salmonella infections in Michigan (1997-2007): assessment using geographic information system.

BACKGROUND: The majority of U.S. disease surveillance systems contain incomplete information regarding socioeconomic status (SES) indicators like household or family income and educational attainment in case reports, which reduces the usefulness of surveillance data for these parameters. We investigated the association between select SES attributes at the neighborhood level and Salmonella infections in the three most populated counties in Michigan using a geographic information system. METHODS: We obtained data on income, education, and race from the 2000 U.S. Census, and the aggregate number of laboratory-confirmed cases of salmonellosis (1997-2006) at the block group level from the Michigan Department of Community Health. We used ArcGIS to visualize the distribution, and Poisson regression analysis to study associations between potential predictor variables and Salmonella infections. RESULTS: Based on data from 3,419 block groups, our final multivariate model revealed that block groups with lower educational attainment were less commonly represented among cases than their counterparts with higher education levels (< high school degree vs. > or = college degree: rate ratio (RR) = 0.79, 95% confidence interval (CI):0.63, 0.99; > or = and high school degree, but no college degree vs. > or = college degree: RR = 0.84, 95% CI: 0.76, 0.92). Levels of education also showed a dose-response relation with the outcome variable, i.e., decreasing years of education was associated with a decrease in Salmonella infections incidence at the block group level. CONCLUSION: Education plays a significant role in health-seeking behavior at the population level. It is conceivable that a reporting bias may exist due to a greater detection of Salmonella infections among high education block groups compared to low education block groups resulting from differential access to healthcare. In addition, individuals of higher education block groups who also have greater discretionary income may eat outside the home frequently and be more likely to own pets considered reservoirs of Salmonella, which increase the likelihood of contracting Salmonella infections compared to their counterparts with lower levels of education. Public health authorities should focus on improving the level of disease detection and reporting among communities with lower income and education and further evaluate the role of higher educational attainment in the predisposition for salmonellosis.

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR.

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.

Antimicrobial susceptibility of Escherichia coli isolates from dairy farms using organic versus conventional production methods.

OBJECTIVE: To compare antimicrobial susceptibility patterns of Escherichia coli isolates cultured from fecal samples from cows and calves on dairy farms that used organic (ie, no or severely limited antimicrobial use) versus conventional production methods. DESIGN: Cross-sectional study. SAMPLE POPULATION: Fecal samples from 10 cows and 10 calves on each of 30 organic dairy farms and 30 neighboring conventional dairy farms in Wisconsin. PROCEDURE: E. coli isolates obtained from the fecal samples were tested for susceptibility to 17 antimicrobials by means of a microbroth dilution test. Prevalence of antimicrobial resistance was compared between organic and conventional dairy farms. RESULTS: E. coli was isolated from 1,121 (94%) fecal samples. Farm type (organic vs conventional) and animal age (cow vs calf) were significantly associated with odds that E. coli isolates would be resistant to various antimicrobials. After controlling for age, logistic regression analyses indicated that isolates from conventional dairy farms had significantly higher rates of resistance to ampicillin, streptomycin, kanamycin, gentamicin, chloramphenicol, tetracycline, and sulfamethoxazole than did isolates from organic dairy farms. However, no significant differences were detected for the 10 other antimicrobials that were tested. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that compared with isolates from conventional dairy farms, E. coli isolates from organic dairy herds have significantly lower prevalences of resistance to 7 antimicrobials; however, prevalence of resistance was not significantly different for 10 other antimicrobials. Resistance was more common for isolates from calves than for isolates from adult dairy cows.

Prevalence and tissue distribution of Besnoitia darlingi cysts in the Virginia opossum (Didelphis virginiana) in Michigan.

Specimens of Virginia opossums (Didelphis virginiana) in Michigan were examined over 1 year to document the presence of Besnoitia darlingi cysts. Cyst morphology, prevalence, seasonal variation, and tissue sites of isolation were studied. Histology and ultrastructural features of the detected cysts and bradyzoites were consistent with B. darlingi. In the opossums, B. darlingi had intracellular tissue cysts. Tissue cysts had a mean diameter of 560 microm and were separated from the host tissue by a thick (5-20 microm) cyst wall. Overall prevalence of B. darlingi cysts in opossums was 10.9% (15/137). Variations in the prevalence were detected during spring (3/17; 17.6%), summer (10/34; 29.4%), and fall (2/60; 3.3%). No cysts were detected in the specimens examined during winter (0/26; 0%). Numerous B. darlingi cysts were detected in ears, conjunctiva, tongue, abdominal muscles, diaphragm, stomach, heart, liver, kidney, lung, and spleen. Cysts were detected mainly in adult female opossums that were debilitated. Ear was the most frequent organ from which the cysts were reported (10/15; 66.7%) when compared individually with other body tissues (P<0.05).

Effects of temperature and host cell type on the in vitro growth and development of Sarcocystis falcatula.

The growth and development of Sarcocystis falcatula in four different cultured cell lines [vero cells, bovine turbinate (BT) cells, equine dermal (ED) cells, and human Hep-2 cells] inoculated with culture-derived merozoites are described. Parasite yields, viability, and plaque forming efficiency were used to compare the growth between different cell lines. Additionally, each cell line was tested at two temperatures of incubation (35 degrees C and 37 degrees C). Based on yield, viability, and plaque forming efficiency, vero cells and BT cells supported growth of S. falcatula better than ED cells and Hep-2 cells. During an 18-day culture period, vero cells produced a mean total of 1.3x10(7) S. falcatula merozoites/T25 flask, BT cells 1.1x10(7), ED cells 0.9x10(7), and Hep-2 cells 0.7x10(7) merozoites/T25 flask. All experimental cell lines grew equally well at 35 degrees C and 37 degrees C. The type of host cells but not the temperature of incubation had a profound effect on the in vitro growth and proliferation of S. falcatula.

Purification of Sarcocystis neurona sporocysts from opossum (Didelphis virginiana) using potassium bromide discontinuous density gradient centrifugation.

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.