Weeks In Phosphate-buffered Saline Research Articles

3D Bioprinting with Visible Light Cross-Linkable Mucin-Hyaluronic Acid Composite Bioink for Lung Tissue Engineering.

3D printing can revolutionize personalized medicine by allowing cost-effective, customized tissue-engineering constructs. However, the limited availability and diversity of biopolymeric hydrogels restrict the variety and applications of bioinks. In this study, we introduce a composite bioink for 3D bioprinting, combining a photo-cross-linkable derivative of Mucin (Mu) called Methacrylated Mucin (MuMA) and Hyaluronic acid (HA). The less explored Mucin is responsible for the hydrogel nature of mucus and holds the potential to be used as a bioink material because of its plethora of features. HA, a crucial extracellular matrix component, is mucoadhesive and enhances ink viscosity and printability. Photo-cross-linking with 405 nm light stabilizes the printed scaffolds without damaging cells. Rheological tests reveal shear-thinning behavior, aiding cell protection during printing and improved MuMA bioink viscosity by adding HA. The printed structures exhibited porous behavior conducive to nutrient transport and cell migration. After 4 weeks in phosphate-buffered saline, the scaffolds retain 70% of their mass, highlighting stability. Biocompatibility tests with lung epithelial cells (L-132) confirm cell attachment and growth, suggesting suitability for lung tissue engineering. It is envisioned that the versatility of bioink could lead to significant advancements in lung tissue engineering and various other biomedical applications.

Melt-Spun, Cross-Section Modified Polycaprolactone Fibers for Use in Tendon and Ligament Tissue Engineering

Tissue Engineering is considered a promising route to address existing deficits of autografts and permanent synthetic prostheses for tendons and ligaments. However, the requirements placed on the scaffold material are manifold and include mechanical, biological and degradation-related aspects. In addition, scalable processes and FDA-approved materials should be applied to ensure the transfer into clinical practice. To accommodate these aspects, this work focuses on the high-scale fabrication of high-strength and highly oriented polycaprolactone (PCL) fibers with adjustable cross-sectional geometry and degradation kinetics applying melt spinning technology. Four different fiber cross-sections were investigated to account for potential functionalization and cell growth guidance. Mechanical properties and crystallinity were studied for a 24-week exposure to phosphate-buffered saline (PBS) at 37 °C. PCL fibers were further processed into scaffolds using multistage circular braiding with three different hierarchical structures. One structure was selected based on its morphology and scaled up in thickness to match the requirements for a human anterior cruciate ligament (ACL) replacement. Applying a broad range of draw ratios (up to DR9.25), high-strength PCL fibers with excellent tensile strength (up to 69 cN/tex) could be readily fabricated. The strength retention after 24 weeks in PBS at 37 °C was 83–93%. The following braiding procedure did not affect the scaffolds’ mechanical properties as long as the number of filaments and the braiding angle remained constant. Up-scaled PCL scaffolds resisted loads of up to 4353.88 ± 37.30 N, whilst matching the stiffness of the human ACL (111–396 N/mm). In conclusion, this work demonstrates the fabrication of highly oriented PCL fibers with excellent mechanical properties. The created fibers represent a promising building block that can be further processed into versatile textile implants for tissue engineering and regenerative medicine.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Persistence of Norwalk virus, male-specific coliphage, and Escherichia coli on stainless steel coupons and in phosphate-buffered saline.

Human noroviruses (NoVs) are a leading cause of acute gastroenteritis and are frequently transmitted by contaminated food, water, hands, and environmental surfaces. Little is known about their environmental stability and/or which alternative microorganisms can serve as effective surrogates. To examine whether Escherichia coli and male-specific coliphage MS2 can be appropriate surrogates for NoVs, approximately 6.8 log genomic equivalent copies of Norwalk virus (NV), and 6.0 to 6.5 log PFU or CFU of MS2 and E. coli, respectively, were inoculated onto stainless steel coupons and held at 4°C, room temperature (RT), or 37°C over a period of 75 min (E. coli and MS2) to 4 weeks. These three microorganisms were also seeded into phosphate-buffered saline (PBS) and sampled at different time intervals for up to 6 weeks. MS2 and E. coli survived approximately 15 min at 37°C, 45 min at RT, and 60 min at 4°C on the stainless steel surfaces. In contrast, NV RNA titers were reduced by only 2.4 log at 37°C, 1.5 log at RT, and 0.9 log at 4°C after 4 weeks. MS2 and E. coli were able to survive at least 5 weeks in PBS at 4°C and RT, and NV was stable in PBS at 4°C and RT for at least 6 weeks. However, E. coli, MS2, and NV were completely inactivated after 1-, 4-, and 5-week incubations in PBS at 37°C, respectively. These findings indicate that NoVs are highly persistent on environmental surfaces and in PBS solution at different temperatures. While E. coli does not appear to be an appropriate surrogate for NoVs, MS2 could be more relevant for modeling the environmental persistence of NoVs under wet conditions, but not under dry conditions.

Preparation and <i>In Vitro</i> Degradation of PLGA/HA Composite Fiber Scaffolds by Electrospinning

The preparation of scaffolds consisted of a polymer composites with inorganic materials has received extensive attention in the field of tissue engineering recently.Electrospinning is a good method to obtain nano-scaled fibers from polymer solutions. In this paper ,we successfully prepared nanofibers of poly(lactide-co-glycolde) (PLGA)/hydroxyapatite(HA)by electrospinning.Their structure morphology and degradation behaviors were studied.The in vitro degradation of PLGA/ HA composite membranes was examined for up to 16 weeks in phosphate-buffered saline (PBS, pH 7.4) at 37°C. The results indicated that the surface of hybrid fibers was much coarser because of the introduction of HA nano-particles. The alkaline HA nano-particles inhibited the self-catalysis of PLGA during the degradation and slowed down the degradation rates of hybrid fibers.The introduction of hydroxyapatite component changed the hydrophilic/hydrophobic balance and, thus, influenced degradation behavior and mechanical properties of the composite membranes during degradation. The preliminary results show that comprehensive properties are well better on the hybrid scaffolds than pure scaffolds and PLGA/ HA bionanocomposites have the potential in tissue regeneration applications.

Premixed acidic calcium phosphate cement: Characterization of strength and microstructure

By using a premixed calcium phosphate cement (CPC), the handling properties of the cement are drastically improved, which is a challenge for traditional injectable CPCs. Previously premixed cements have been based on apatitic cements. In this article, acidic cement has been developed and evaluated. Monocalcium phosphate monohydrate and beta-tricalcium phosphate were mixed with glycerol to form a paste. As the paste does not contain water, no setting reaction starts and thus the working time is indefinite. Powder/liquid ratios (P/L) of 2.25, 3.5 and 4.75 were evaluated. Setting time (ST) and compressive strength (CS) were measured after 1 day, 1 week and 4 weeks in phosphate buffered saline (PBS) solution, and the corresponding microstructure was evaluated using electron microscopy and X-ray diffraction. The ST started when the cements were placed in PBS and ranged from 28 to 75 min, higher P/L gave a lower ST. Higher P/L also gave a higher CS, which ranged from 2 to 16 MPa. The microstructure mainly consisted of monetite, 1-5 microm in grain size. After 4 weeks in PBS, the strength increased. As acidic cements are resorbed faster in vivo, this cement should allow faster bone regeneration than apatitic cements. Premixed cements show a great handling benefit when compared with normal CPCs and can be formulated with similar ST and mechanical properties.

Accelerated Conversion of Silicate Bioactive Glass (13-93) to Hydroxyapatite in Aqueous Phosphate Solution Containing Polyanions

The influence of alginic acid, an anionic polysaccharide, on the conversion of 13-93 bioactive glass to hydroxyapatite (HA) in aqueous phosphate solution was investigated. Disks of 13-93 glass were immersed in phosphate-buffered saline (PBS) containing 0-1 weight percent (wt%) alginic acid. HA was detected on the glass surface by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy after 2 weeks in PBS containing 0.5 wt% alginic acid, compared with 6 weeks in PBS without alginic acid. In the presence of alginic acid (0.5 wt%), the growth of the HA layer on the glass increased almost linearly with time, at a rate that was approximately six times faster than that for the system without alginic acid. The thickness of the HA layer increased with the concentration of alginic acid (0-1 wt%). Chelation of the electron-donating moieties of alginic acid polymer, such as carboxyls and hydroxyls, to the leached cations from the bioactive glass, in particular calcium ions, is suggested as a mechanism for the enhanced corrosion of the glass and, hence, the enhanced conversion to HA. The use of natural or synthetic polyelectrolytes could provide a method for manipulating the conversion rate of bioactive glass to HA.

MBG/PLGA composite microspheres with prolonged drug release

Mesoporous bioactive glass (MBG) and composite microspheres with MBG particles embedded in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) matrix have been prepared and used to load gentamicin (GS). The in vitro drug release experiments from both MBG and composite microspheres were conducted in distilled water and phosphate buffered saline (PBS) solution at 37 degrees C for more than 30 days. In both water and PBS, GS release from the MBG was very fast with about 60 wt % of the loaded drug released in the first 24 h, and more than 80 wt % released in two days. MBG/PLGA composite microspheres showed an initial release of about 33 wt % in the first day, and 48 wt % in 2 days, and a subsequent sustained release lasting for more than 4 weeks in PBS. MBG/PLGA composite microspheres may be used as an alternative drug release system, especially as a bone void filler for bone repair due to their combined advantages of sustained release of antibiotics and apatite-forming ability.

The Compressive Properties of Bone Cements Containing Large Doses of Antibiotics

The addition of large amounts of antibiotics to bone cement provides a convenient local delivery, but may influence the compressive properties of the cement. Flucloxacillin and vancomycin were added to Simplex P (Stryker, Limerick, Ireland) and VersaBond (Smith & Nephew) cements. Tripling the antibiotic dose from 2 to 6 g had little effect on the static compressive properties 24 hours after curing. After 4 weeks in phosphate-buffered saline, there was marked decrease in properties with the addition of antibiotics. Compressive strength of cements with 6 g of antibiotic was reduced to near or below the ASTM and ISO minimum of 70 MPa after 4 weeks in phosphate-buffered saline. Microcomputer tomography revealed increased porosity and clumping of the radiopacifier with the addition of antibiotics.

In Vitro and Intracellular Production of Peptide‐Encapsulated Fluorescent Silver Nanoclusters

In Vitro and Intracellular Production of Peptide‐Encapsulated Fluorescent Silver Nanoclusters

Degradation of electrospun PLGA-chitosan/PVA membranes and their cytocompatibility in vitro

Nanofibrious composite poly(lactide-co-glycolide) (PLGA) and chitosan/poly(vinyl alcohol) (PVA) membranes were prepared by simultaneously electrospinning PLGA and chitosan/PVA from two different syringes. The in vitro degradation of PLGA and cross-linked composite membranes was examined for up to 10 weeks in phosphate-buffered saline (PBS, pH 7.4) at 37°C. The pH of PBS, the weight average molecular weight of PLGA, fiber morphology and mechanical properties, including tensile strength, Young's modulus and elongation-at-break, were measured as a function of degradation time. The fibrous composite membranes were further investigated as a promising scaffold for human embryo skin fibroblasts (hESFs) culture. The cell adhesion and morphology of hESFs seeded on each electrospun membrane was observed using scanning electron microscope and inverted phase contrast microscopy after Wright–Giemsa staining. The introduction of chitosan/PVA component changed the hydrophilic/hydrophobic balance and, thus, influenced degradation behavior and mechanical properties of the composite membranes during degradation. The cells could not only favorably attach and grow well on the composite membranes, but were also able to migrate and infiltrate the membranes. Therefore, the results suggest that the composite membranes can positively mimic the structure of natural extracellular matrices and have the potential for application as three-dimensional tissue-engineering scaffolds.

Hydrolytic degradation of porous scaffolds for tissue engineering from terpolymer of l-lactide, ε-caprolactone and glycolide

Segmental terpolymer of l-lactide, ε-caprolactone and glycolide have been synthesized by ring-opening polymerization with the use of zirconium acetylacetonate as a biocompatible initiator. Porous scaffolds aimed at tissue engineering have been prepared from resulting terpolymer by solvent casting/particulate leaching technique. Sieved sodium citrate particles of 500–700 μm size have been used as porogens. The obtained scaffolds have been submitted to degradation in phosphate buffered saline (PBS) at 37 °C for 26 weeks and characterized as a function of incubation time by: Fourier transform infrared spectroscopy in the attenuated total reflection mode (FTIR-ATR), nuclear magnetic resonance ( 1H NMR), gel permeation chromatography (GPC) and scanning electron microscopy (SEM). The scaffolds start to degrade immediately after contact with PBS, as demonstrated by decrease in molecular weights ( M n and M w). In the first stage (6 weeks in PBS) when the scaffolds are still dimensionally stable, the degradation is generally due to cleavage of ester bonds between glycolidyl (GG) and caproyl (Cap) groups present in chain sequences such as –GGCapCap–, –GGGCap–, as shown by 1H NMR. The analysis of FTIR spectra of the initial scaffolds and after 6 week immersion in PBS reveals a shift of the band attributed to C O vibrations and considerable changes in the shape of the bands attributed to C–O and C–O–C vibrations. These changes are due to chain-scission of polyester bonds in hydrolysis reaction. Subsequently, when the concentration of sequences containing glycol, glycolidyl and caproyl groups decreases, the influence of cleavage of polyester bonds between longer lactyl microblocks on the degradation increases gradually. As a result, two separate polymer fractions are formed.

Membrane-encased polymer millirods for sustained release of 5-fluorouracil.

This article describes the design and development of a novel membrane-encased polymer millirod for the sustained release of an anticancer drug, 5-fluorouracil (5-FU). The millirod consists of two functional compartments: (1) an inner 5-FU-loaded monolithic millirod as the drug depot, and (2) an outer NaCl-impregnated polymer membrane to control the release rate of 5-FU. The inner millirod is fabricated by a compression-heat molding procedure to permit the entrapment of 5-FU particles in the poly(D,L-lactide-co-glycolide) (PLGA) matrix. The drug loading density is controlled at 30 w/w% to achieve a burst release of 5-FU (>90% of the drug are released within 48 h) from the monolithic millirod. The NaCl-impregnated PLGA membrane is generated by solvent casting and is then wrapped over the monolithic millirod to produce the membrane-encased millirod. Scanning electron microscopy shows that dissolution of NaCl particles produces a semipermeable polymer membrane to provide a sustained release of 5-FU. The membrane thickness and the density of NaCl particles inside the membrane are useful parameters to control the release kinetics of 5-FU. Under the experimental conditions in this study, sustained release of 5-FU [rates between 0.1 and 0.4 mg/(day. cm of millirod)] is achieved for 2 to 5 weeks in phosphate-buffered saline (pH 7.4) at 37 degrees C. Results from this study demonstrate that membrane-encased polymer millirods provide controllable sustained release kinetics for applications in intratumoral drug delivery.

Physical and metabolic causes of sub-lethal damage in Listeria monocytogenes after long-term chilled storage at 4 degrees C.

Cells of Listeria monocytogenes display sub-lethal injury when subjected to long-term chill-storage in a nutrient-poor environment. The physical and metabolic causes of sub-lethal injury to two meat (L61 and L62) and two clinical (L98 and L99) L. monocytogenes strains chill-stored (4 degrees C) for 4 weeks in phosphate-buffered saline at pH 7.0 and pH 5.5, and pH 5.5 in the presence of 0.3% potassium sorbate, were characterized. Causes of sub-lethal injury were determined by examining changes in the cell structure, leakage of nucleic acids and proteins from the cells, and cell recovery from injury in the presence of the metabolic inhibitors rifampicin, D-cycloserine, carbonyl cyanide m-chlorophenylhydrazone and chloramphenicol. Visible shrinkage of the cytoplasm and slight cell wall damage were apparent over the 4 week storage period by electron microscopy for all four strains and three storage conditions. By contrast, over the same storage period, only three of the strains (L62, L98 and L99) displayed slight leakage of cellular content in all three storage media, while one strain (L61) displayed greater leakage. The three strains also displayed similar storage media-dependent metabolic damage. For these strains, phosphate-buffered saline pH 5.5 caused the least damage and potassium sorbate, the most. Recovery experiments also indicated that at pH 5.5, the energy transduction system of these three strains remained undamaged, and that injury to the cell transcription machinery was greatest at pH 7.0. The fourth strain, L61, displayed less damage than the others but this was attributed to the death of the injured cell sub-population in this strain. In this study, damage to sub-lethally injured chill-stored L. monocytogenes was different from that caused by other agents, such as heat. Therefore, cells injured by chill-storage under starvation conditions may require novel protocols to assure their effective recovery.

Growth and survival of Bordetella bronchiseptica in natural waters and in buffered saline without added nutrients

Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.

Cell Flow Cytometry of Fixed and Unfixed Bursal and Thymic Cells

Analysis in a cell flow cytometer (Ortho Spectrum III, Ortho Inst.) of single cell suspensions from the bursa and thymus of 20-day embryos revealed two distinct cell clusters. The two clusters were less apparent after fixation of the cells in paraformaldehyde and assumed a comet-like appearance at 30 min fixation in ethyl alcohol (EA). The G (postmitotic), S [deoxyribonucleic acid (DNA) synthesis], and G2 /M (premitotic and mitotic) phases of the life cycle were visualized in two cell flow cytometers (Ortho Spectrum III and FACS IV, Bectin Dickinson) after treating the cells with EA, ribonuclease (RNase), and propidium iodide (PI, a fluorescent dye). Bursal cell suspensions exposed to the EA-RNase-PI protocol and stored for 3 weeks in phosphate-buffered saline showed minor changes in the G1 coefficient of variation, G1, and S percentages, but marked changes in these parameters occurred after 6 weeks of storage. Thymic cells treated in a similar fashion could not be maintained for 3 weeks. Bursal and thymic cells may remain in the EA for one day and possibly as long as 7 days prior to preparing them for DNA life cycle analysis. Paraformaldehyde was not a satisfactory cell fixative for assessing a cell's DNA life cycle.

Effects of fixation for immunohistochemistry on tissue concentrations of substance P and somatostatin determined by radioimmunoassay.

Abstract Concentrations of substance P and somatostatin were measured in preparations of the myenteric plexus (plus longitudinal muscle) of the guinea-pig ileum after fixation and processing for immunohistochemistry and compared with concentrations measured in fresh tissue. Two fixative solutions were used: (i) 4% formalin in phosphate buffer (0.1 M, pH 7.0); and (ii) a mixture of aqueous picric acid with 2% formalin in phosphate buffer (0.1 M, pH 7.0). Tissues were extracted in boiling aqueous acetic acid (2.0 M) either immediately after fixation and processing or after storage for up to four weeks in phosphate-buffered saline (PBS) with or without sodium azide. The concentrations of substance P and somatostatin in these extracts were measured by radioimmunoassay and compared to the concentrations in extracts of fresh tissue. The concentration of substance P in fixed tissue was the same as that found in fresh tissue, whereas the concentration of somatostatin in fixed tissue was half that found in fresh tissue ( P