Direct Contact Assay Research Articles

Evaluation of the antifungal activity and modulation between Cajanus cajan (L.) Millsp. leaves and roots ethanolic extracts and conventional antifungals

Background:The use and investigation of natural products with antimicrobial activity from vegeral source have been reported by several researchers. Cajanus cajan (Fabaceae) is a multiple use specie mainly as human food. In popular medicine, diverse parts of the plant are used as sedative and to treat cough, hepatitis, and diabetes.Materials and Methods:This study shows the characterization of secondary metabolites present in ehtanolic extracts from leaves and roots of Cajanus cajan by phytochemical prospection. The evaluation of the antifungal activity was performed by the microdilution method, and from the subinhibitory concentrations (MIC 1/8) the modulatory activity of antifungical (fluconazole and ketoconazole) was analyzed by the direct contact assay against C. albicans ATCC40006, Candida krusei ATCC 6538 and Candida tropicalis ATCC 40042.Results:The results showed the presence of tannins, flavonoids, and alkaloids in both extracts as the clinically relevant antifungal activity. The modulatory potential is presented by the antifungal tested against yeasts.Conclusion:The extracts studied here have demonstrated to be a new therapeutic source to treat these microorganism-associated diseases.

Antifungal Activity of Essential Oil from Asteriscus graveolens against Postharvest Phytopathogenic Fungi in Apples

The essential oils of the aerial parts of Asteriscus graveolens have been studied using GC and GC-MS. Twenty-eight compounds were identified in the essential oil amounting to 94.9% of the total oil. The aerial part oils showed similar chromatographic profiles and were characterized by having a high content of oxygenated sesquiterpenes with 6-oxocyclonerolidol (66.7% +/- 5.5) and 6-hydroxycyclonerolidol (8.8% +/-1.2) as the major components. The antifungal effect of the essential oil from A. graveolens leaves was evaluated in vitro against three phytopathogenic fungi of apples (Alternaria sp., Penicillium expansum, and Rhizopus stolonifer). The results suggest that this essential oil has fungicidal properties towards Alternaria sp. from direct contact assay at 0.2% (v/v) and to P. expansum from vapor assay tests at 80 microL.

Portland cement for bone tissue engineering: Effects of processing and metakaolin blends

The need for a suitable scaffolding material for load bearing bone tissue engineering still has yet to be met satisfactorily. In this study, Portland cement and Portland cement/metakaolin (MK) blends were processed to render them biologically and mechanically suitable for such application. Portland cement was mixed with MK at different ratios. The slurries were hydrated under atmospheric (noncarbonated samples) and high-CO₂ conditions (carbonated samples). The mechanical properties were characterized via compressive tests. The bioactivity was analyzed in a simulated body fluid solution. Scanning electron microscopy and energy dispersive spectroscopy were used to evaluate sample morphology and chemistry. The cytocompatibility (direct contact assay, MTT test, and alkaline phosphatase activity) was tested using human osteoblast-like cells. Cell responses were observed via conventional and electron microscopy. The results showed that the implementation of MK did not significantly influence the mechanical properties. All the samples evidenced bioactive behavior. Cell experiments confirmed a highly cytotoxic response to the noncarbonated specimens. The introduction of MK as well as the CO₂ pretreatment significantly improved the cytocompatibility of the specimens. These results show that properly processed Portland cement and Portland cement/MK blends could present suitable properties for the development of load-bearing scaffolding structures in bone tissue-engineering applications.

A Study of the Cytotoxicity of a New Nonwoven Polymeric Fibrous Bandaging Material In-Vitro

Traditionally used cotton-based bandaging materials have several disadvantages which can be overcome by using another fabric structure – nonwoven fabric. Moreover, these materials are more spongeous which increases their sorption capacity. The new bandaging material developed by the Institute of Physics of Strength and Material Science of the Siberian Branch of the Russian Academy of Sciences has even better sorption capacity with improved sorption properties. Its sorption capacity has been increased by means of an additional introduction of porous aluminum hydrate particles into fabric. It is important that a bandaging material has a good biocompatibility and does not have any cytotoxic effect on cells and tissues. Here it is present results of the study of the material's direct contact and indirect cytotoxicity assays in comparison with cotton gauze. It have found that in the direct contact of nonwoven polymeric fibrous bandaging material (NPFBM) with cells for 24 hours of cultivation no changes in cell morphology take place, nor does the amount of dead cells increase. These conclusions have been made by means of both a visual examination and an МТТ assay. The NPFBM extract did not have any cytotoxic effect on the tested cells either. The obtained results allow us to make a conclusion that the NPFBM complies with the international standard ISO 10993-5, which is applied to medical goods, and can from now on be applied in the treatment of infected wounds in clinical practice.

The Use of Zein and Shuanghuangbu for Periodontal Tissue Engineering

AimTissue engineering is a promising area with a broad range of applications in the fields of regenerative medicine and human health. The emergence of periodontal tissue engineering for clinical treatment of periodontal disease has opened a new therapeutic avenue. The choice of scaffold is crucial. This study was conducted to prepare zein scaffold and explore the suitability of zein and Shuanghuangbu for periodontal tissue engineering.MethodologyA zein scaffold was made using the solvent casting/particulate leaching method with sodium chloride (NaCl) particles as the porogen. The physical properties of the zein scaffold were evaluated by observing its shape and determining its pore structure and porosity. Cytotoxicity testing of the scaffold was carried out via in vitro cell culture experiments, including a liquid extraction experiment and the direct contact assay. Also, the Chinese medicine Shuanghuangbu, as a growth factor, was diluted by scaffold extract into different concentrations. This Shuanghuangbu-scaffold extract was then added to periodontal ligament cells (PDLCs) in order to determine its effect on cell proliferation.ResultsThe zein scaffold displayed a sponge-like structure with a high porosity and sufficient thickness. The porosity and pore size of the zein scaffold can be controlled by changing the porogen particles dosage and size. The porosity was up to 64.1%–78.0%. The pores were well-distributed, interconnected, and porous. The toxicity of the zein scaffold was graded as 0–1. Furthermore, PDLCs displayed full stretching and vigorous growth under scanning electronic microscope (SEM). Shuanghuangbu-scaffold extract could reinforce proliferation activity of PDLCs compared to the control group, especially at 100 μg·mL−1 (P<0.01).ConclusionA zein scaffold with high porosity, open pore wall structure, and good biocompatibility is conducive to the growth of PDLCs. Zein could be used as scaffold to repair periodontal tissue defects. Also, Shuanghuangbu-scaffold extract can enhance the proliferation activity of PDLCs. Altogether, these findings provide the basis for in vivo testing on animals.

Melt Processing of Chitosan‐Based Fibers and Fiber‐Mesh Scaffolds for the Engineering of Connective Tissues

We report the production of chitosan-based fibers and chitosan fiber-mesh structures by melt processing (solvent-free) to be used as tissue-engineering scaffolds. The melt-based approach used to produce the scaffolds does not change their main characteristics, including the surface roughness and microporosity. The porosity, pore size, interconnectivity and mechanical performance of the scaffolds are all within the range required for various tissue-engineering applications. Biological assessments are performed in direct-contact assays. Cells are able to colonize the scaffold, including the inner porous structure. The cells show high indices of viability in all of the scaffold types.

Biological evaluation of a novel copper-containing composite for contraception

To investigate the biocompatibility of a novel copper-containing composite to provide preclinical data for clinical application of intrauterine device (IUD) or intra-vas device (IVD). Prospective experimental study. Good laboratory practices laboratories. Twenty healthy adult mice (SPF grade Kunming white mice, animal code SCXK 2003-0005). Cytotoxicity tests in vitro were conducted to evaluate the influence of the materials on the morphology, growth, and proliferation of cultured L929 mouse fibroblasts. Acute systemic toxicity tests were conducted to investigate the acute systemic toxic reaction with mice, and then the materials were implanted into the spinal muscle of rabbits (n = 15). The rabbits were sacrificed for pathologic examination at 1, 4, and 12 weeks after surgery. Evaluation of cytotoxicity by MTT assay, cytotoxicity test by direct contact assay, acute systemic toxicity test, and material implantation test. The cytotoxicity grade of the copper-containing composite was 0-1, suggesting that the material was free of cytotoxicity; no acute systemic toxicity was found in any mice; mild inflammatory reaction was observed in the surrounding tissues of the implanted material in the early implantation stage, which was similar to that of the sham-operated sides. Twelve weeks after implantation, the inflammatory reaction was completely disappeared in the implanted tissue, similarly to the sham-operated sides. The fibrosis membrane surrounding the material became stable gradually over time. The copper-containing composite has excellent biocompatibility, which is feasible and safe for the clinical application as a novel contraceptive material.

Injection Molding of Titanium Alloy Implant For Biomedical Application Using Novel Binder System Based on Palm Oil Derivatives

Problem statement: Titanium alloy (Ti6Al4V) has been widely used as an implant for biomedical application. In this study, the implant had been fabricated using high technology of Powder Injection Molding (PIM) process due to the cost effective technique for producing small, complex and precision parts in high volume compared with conventional method through machining. Approach: Through PIM, the binder system is one of the most important criteria in order to successfully fabricate the implants. Even though, the binder system is a temporary, but failure in the selection and removal of the binder system will affect on the final properties of the sintered parts. Therefore, the binder system based on palm oil derivative which is palm stearin had been formulated and developed to replace the conventional binder system. Results: The rheological studies of the mixture between the powder and binders system had been determined properly in order to be successful during injection into injection molding machine. After molding, the binder held the particles in place. The binder system had to be removed completely through debinding step. During debinding step, solvent debinding and thermal pyrolysis had been used to remove completely of the binder system. The debound part was then sintered to give the required physical and mechanical properties. The in vitro biocompatibility also was tested using Neutral Red (NR) and mouse fibroblast cell lines L-929 for the direct contact assay. Conclusion: The results showed that the properties of the final sintered parts fulfill the Standard Metal Powder Industries Federation (MPIF) 35 for PIM parts except for tensile strength and elongation due to the formation of titanium carbide. The in vitro biocompatibility on the extraction using mouse fibroblast cell line L-929 by means of NR assays showed non toxic for the sintered specimen titanium alloy parts.

Cell Adhesion and Proliferation onto Chitosan-based Membranes Treated by Plasma Surface Modification

Surface properties play a vital role in the functioning of a biomaterial. Cellular adherence and growth onto biomaterials can be enhanced in biomaterial modifications of their surface. In this work, the cell behavior on chitosan membranes modified by argon and nitrogen-plasma treatments was investigated. Characterization of the membranes was performed using atomic force microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Cytotoxicity assessment and direct contact assay were carried out for untreated and treated chitosan membranes using L929 fibroblast-like cells. Cell morphology and cell viability were assessed to evaluate the cell attachment and proliferation. Changes in terms of roughness, surface chemistry, and hydrophilicity/hydrophobic balance of chitosan-modified membranes were observed. Regarding cell studies, the findings revealed that the extracts of all membranes do not induce cytotoxic effects. Moreover, the in vitro assays evidenced an improvement of the L929 adhesion and attachment when compared to untreated chitosan membranes. Overall, the data obtained clearly demonstrated that plasma treatments constitute an effective way of improving the biocompatibility of chitosan membranes towards to their use in biomedical applications.

Impact of assay selection and study design on the outcome of cytotoxicity testing of medical devices: The case of multi-purpose vision care solutions

Medical device biocompatibility testing usually includes a cytotoxicity component. Assay selection and protocol design often depend on a specific testing standard rather than on the characteristics of the medical device. To better understand the impact of assay selection on study outcome of unstructured medical devices, we evaluated contact lens multi-purpose solutions (MPS) in the agar diffusion, direct contact and two elution cytotoxicity assays. To simulate the conditions of use, MPS were evaluated alone and in combination with contact lenses. All MPS passed the agar diffusion assay (n=3) and extracts prepared from contact lenses soaked in MPS passed the USP elution assay (n=3). Both the duration of contact and MPS concentration impacted the outcome of a modified elution assay. When tested at 25% strength for 48h, all MPS evaluated were non-cytotoxic (n>3). Test article movement and mechanical damage were significant issues with the direct contact assay. Movement was effectively controlled by manipulating contact lens orientation while using 0.8mL culture medium. All MPS passed the USP direct contact cytotoxicity test when evaluated using this optimized methodology (n=3). These data are consistent with MPS results in ocular irritation studies in rabbits (n=3).

Chemical Composition and Antibacterial Evaluation of the Essential Oil of Zanthoxylum articulatum

The essential oil of the aerial parts of Zanthoxylum articulatum was obtained by hydrodistillation and examined by GC-MS. The oil was tested against Gram negative (Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 10536) and Escherichia coli (EC 27) and Gram positive (Staphylococcus aureus (ATCC 12692) and Staphylococcus aureus (SA 358)) bacteria at different concentrations using microdillution and gel diffusion methods. The synergistic activity between this essential oil and aminoglycosides were analyzed by direct contact and gaseous contact assays. The essential oil showed significant antibacterial activity particularly against S. aureus (ATCC12692) and the multiresistant S. aureus strain (SA 358) with inhibition halos of 17.00 ± 5.60 and 13.50 ± 4.90, respectively. A clinical and relevant inhibitory activity was observed against S. aureus (SA 358), with a minimum inhibitory concentration (MIC) value of 256 μg/mL. The results demonstrated the synergism between the essential oil and the aminoglycosides amikacin, tobramycin and gentamicin against S. aureus (SA 358) and only gentamicin didn’t show synergism against P. aeruginosa (ATCC 15442), but the antagonism wasn’t verified too.

New direct contact approach to evaluate soil genotoxicity using the Vicia faba micronucleus test

A method to assess micronucleus (MN) induction in Vicia faba roots by direct contact exposure to a solid matrix was developed. The procedure comprised a 5-d germination period, as in the well-known method using aqueous extracts. However, the seeds were here sown directly into the test soil whereas a culture period is necessary before exposing seedlings to a liquid medium. One soil under forest and two contaminated soils from areas affected by industrial installations and a coke works were used. Three durations of direct exposure were tested: 2, 5 and 7 d. The optimal duration was evaluated at 2 d to observe maximal MN induction without observing toxicity symptoms. The methodology using aqueous extracts was applied to the same three soils: MN frequency was higher than in the direct contact assay but the ratios of MN frequencies from tested soils in comparison to the negative control were lower. However, for each soil, both the direct contact method and the aqueous extract exposure led to the same risk assessment diagnosis. The evaluation of a concentration range of a polycyclic aromatic hydrocarbons (PAH)-contaminated soil showed a dose-dependent MN frequency when the seeds were allowed to germinate before sowing in the soil: the soil genotoxicity was the highest at intermediate doses. The direct contact method was found to be rapid, sensitive and well suited to the evaluation of soil quality.

Surface modification of starch based biomaterials by oxygen plasma or UV-irradiation

Radiation is widely used in biomaterials science for surface modification and sterilization. Herein, we describe the use of plasma and UV-irradiation to improve the biocompatibility of different starch-based blends in terms of cell adhesion and proliferation. Physical and chemical changes, introduced by the used methods, were evaluated by complementary techniques for surface analysis such as scanning electron microscopy, atomic force microscopy, contact angle analysis and X-ray photoelectron spectroscopy. The effect of the changed surface properties on the adhesion of osteoblast-like cells was studied by a direct contact assay. Generally, both treatments resulted in higher number of cells adhered to the modified surfaces. The importance of the improved biocompatibility resulting from the irradiation methods is further supported by the knowledge that both UV and plasma treatments can be used as cost-effective methods for sterilization of biomedical materials and devices.

Designing Supermacroporous Cryogels Based on Polyacrylonitrile and a Polyacrylamide–Chitosan Semi-interpenetrating Network

Cryogels of polyacrylonitrile (PAN) and a semi-interpenetrating network of polyacrylamide–chitosan (PAAC) synthesized at sub-zero temperature have large interconnected pores in the range of 10–100 μm as analyzed by scanning electron microscopy (SEM) and mercury porosimetry with porosity more than 90%. They had good transport properties with a diffusion constant of 3.5 × 10−7 cm2/s for bovine serum albumin and hydraulic permeability of 4 × 10−12 m4/N s. The materials have a high surface area of 9–18 m2/g which is significantly larger than the surface area available in commercially available hollow fiber bioreactors. The cryogels are thermally stable and have good mechanical strength with a Young's modulus of 137–1967 kPa for different concentration of PAN gels and 13 kPa for PAAC cryogel. The cryogels are biocompatible as confirmed by fetal bovine serum protein adsorption and direct contact assay. The material demonstrated adherence and proliferation of cells on the polymer surface and a sustained growth of cells over the scaffolds was seen for a period of 14 days. The three-dimensional (3-D) cryogel network with these physical characteristics renders them as potential material for cell scaffold in a perfusion bioreactor.

Assessment of the Suitability of Chitosan/PolyButylene Succinate Scaffolds Seeded with Mouse Mesenchymal Progenitor Cells for a Cartilage Tissue Engineering Approach

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds' overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (C-PBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 x 5 x 5 mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 mum, retrieving an average pore size of 251.28 mum. Regarding the mechanical properties, the compressive modulus was of 1.73 +/- 0.4 MPa (E(sec) 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow-derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 x 10(5) cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.

Plasma Surface Modification of Chitosan Membranes: Characterization and Preliminary Cell Response Studies

Surface modification of biomaterials is a way to tailor cell responses whilst retaining the bulk properties. In this work, chitosan membranes were prepared by solvent casting and treated with nitrogen or argon plasma at 20 W for 10-40 min. AFM indicated an increase in the surface roughness as a result of the ongoing etching process. XPS and contact angle measurements showed different surface elemental compositions and higher surface free energy. The MTS test and direct contact assays with an L929 fibroblast cell line indicated that the plasma treatment improved the cell adhesion and proliferation. Overall, the results demonstrated that such plasma treatments could significantly improve the biocompatibility of chitosan membranes and thus improve their potential in wound dressings and tissue engineering applications.

The Effect of Chitosan on the In Vitro Biological Performance of Chitosan−Poly(butylene succinate) Blends

Chitosan blends with synthetic biodegradable polymers have been proposed for various biomedical applications due to their versatile mechanical properties and easier processing. However, details regarding the main surface characteristics that may benefit from the blending of these two types of materials are still missing. Hence, this work aims at investigating the surface properties of chitosan-based blends, illustrating the way these properties determine the material-proteins interactions and ultimately the behavior of osteoblast-like cells. The surface characteristics of modified and nonmodified blends were assessed using complimentary techniques such as optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), contact angle measurements and surface energy calculations. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HFN) onto the different surfaces was quantified by association of an indirect method with a colorimetric assay. It was found that the presence of chitosan on the surface promoted the adsorption of proteins. Moreover, a preferential adsorption of albumin over fibronectin was registered. The in vitro biological performance of the studied materials was further investigated by a direct contact assay with an osteoblastic-like cell line (SaOs-2). A synergistic effect of the two components of the blend was observed. While the synthetic polyester promoted the adhesion of SaOs-2, the presence of chitosan significantly enhanced the osteoblastic activity of these cells. This work further confirmed the interest in designing polymeric blends with natural polymers as a successful strategy to enhance the biological performance of a biomaterial.

Synthesis and viscoelastic characterization of novel hydrogels generated via photopolymerization of 1,2-epoxy-5-hexene modified poly(vinyl alcohol) for use in tissue replacement

Hydrogels have been proposed as candidates for tissue replacement; however, current systems are often highly susceptible to hydrolytic degradation and have not been shown to mimic the viscoelastic behavior of the native tissue when subjected to dynamic loading conditions. In the present work, 1,2-epoxy-5-hexene modified poly(vinyl alcohol) was crosslinked via photopolymerization to generate non-degradable hydrogels with mechanical properties and network characteristics that could be modulated through variation in the type and percentage of a monomeric additive. Complex shear moduli obtained from dynamic frequency sweeps in torsional shear were used to exemplify the differences in the viscoelastic behavior of the materials, and the corresponding changes in crosslink density were determined by rubber elasticity theory. Hydrolysis resistance was assessed by monitoring variations in the moduli of hydrogels submerged in Hank’s balanced salt solution for progressively longer periods of time. Over the time-frame of the experiment, no change in the viscoelastic behavior was observed. Direct contact assays and elution tests were used to demonstrate that the system was non-cytotoxic. This study represents a successful attempt to generate a non-degradable hydrogel system with viscoelastic behavior that can be readily modulated to match that of soft biological tissues for use in tissue replacement.

Biocompatibility, Bioactivity and Mechanical Properties of Portland Cement and Portland Cement-Metakaolin Blends for Bone Tissue Engineering Applications

AbstractWe studied the potential applications of Portland cement and Portland cement-Metakaolin blends as scaffolding materials for load bearing bone tissue engineering. Cementitious pastes were prepared by mixing Portland cement and Metakaolin at different ratios (100:0, 85:15), and hydrated under a concentrated CO2 atmosphere (carbonated pastes). Pastes fabricated similarly, but hydrated under normal atmospheric conditions were used for comparison (non-carbonated pastes). Compressive tests were run to evaluate the mechanical properties of the pastes. The bioactivity of the samples was tested in a simulated body fluid (SBF) solution for 1 and 4 days. Sample morphology and chemistry were characterized via scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), respectively. The cytocompatibility was studied using human osteosarcoma (HOS) cell cultures and the direct contact assay. Mechanical characterization did not show significant differences in the compressive strength of the blends compared to pure cement controls. The bioactivity test revealed that the pastes induced surface precipitation of calcium phosphate (CaP) when exposed to the SBF solution (as confirmed by SEM and EDS). Non-carbonated pastes induced early CaP precipitation. Cytocompatibility experiments showed that the carbonated blends allowed adequate cell growth. Non-carbonated blends presented a highly cytotoxic behavior. The introduction of Metakaolin did not affect the cytocompatibility of the samples. These results show that Portland cement and Portland cement-Metakaolin blends could present suitable characteristics for applications as scaffolding materials in load bearing bone tissue engineering.

Antifungal Activity of the Volatile Phase of Essential Oils: A Brief Review

Interest in the antifungal activity of essential oils has increased markedly in recent years. The volatile (vapour) components of several essential oils have been demonstrated to have potent antifungal activity, often in excess of that displayed in direct contact assays. A lack of consistent methodology and reporting, however, hinders direct comparison of publications. A variety of mechanisms have been suggested for the activity of these active volatiles against hyphate fungi. This paper briefly reviews some of the more recent data and identifies areas that require standardization and further study.