Combination Of Alginate Research Articles

Formulation and In vitro Characterization of Floating Gel Beads of Metformin Hydrochloride

A floating type dosage form, gel beads of metformin hydrochloride was prepared by emulsification gelation technique. The gel bead was formed by mixing the polymer in water, oil phase and it was extruded in the calcium chloride solution as curing agent. The formulation parameters optimized were polymer ratio, concentration of oil, curing time on drug content, floating lag time, morphology, swelling of beads and release kinetics.The scanning electron photomicrographs revealed morphology of beads. The size of beads was measured. Entrapment efficiency of drug loaded beads was found to be over 90%. In vitro release of metformin hydrochloride from alginate–pectin beads into simulated gastric fluid at 37 ºC showed no significant burst effect. The cumulative release reached above 74.71 ± 4.15% in about 12h. The use of sodium alginate and combinations of sodium alginate with pectin were used to study the effect on the sustained release of the drug from the formed beads. It was found that sodium alginate was not sufficient to sustain the drug release at gastric pH (fed condition). Appropriate combination of alginate and pectin could provide the sustained release of drug. Floating gel beads formulation provides an alternative delivery for metformin in diabetes treatment.

Rejection of pharmaceuticals by nanofiltration (NF) membranes: Effect of fouling on rejection behaviour

The aim of this study was to investigate the effects of membrane fouling by sodium alginate, latex and a combination of alginate+latex on the rejection behaviour of salts and organics. Sodium chloride and caffeine were selected to represent salts and organics, respectively. The effects of the presence of calcium chloride on the fouling behaviour and rejection of solutes were investigated. The results revealed that the salt rejection by virgin membranes was 47% while that of caffeine was 85%. Fouling by alginate, latex and combined alginate–latex resulted in flux decline of 25%, 37% and 17%, respectively. The addition of Ca2+ aggravated fouling and resulted in further flux decline to 37%. Fouling decreased salt rejection, an observation that was further aggravated by the addition on Ca2+. However, it was also observed that fouling with alginate and calcium and with latex and calcium minimised salt rejection by 30% and 31%, respectively. This reduction in salt rejection was attributed to the decrease in permeate flux (since rejection is a function of flux). There was a slight increase in caffeine rejection when the membrane was fouled with latex particles. Moreover, the presence of foulants on the membrane resulted in a decrease in the surface charge of the membrane. The results of this study have shown that the NF 270 membrane can be used to treat water samples contaminated with caffeine and other organic compounds that have physicochemical properties similar to those of caffeine.

Optimization of Vasculogenesis within Naturally-Derived, Biodegradable Hybrid Hydrogel Scaffolds

INTRODUCTION: Cellular ingrowth and neovascularization of acellular tissue-regeneration scaffolds represent the rate-limiting steps of permanent integration.1 In previous work, we have demonstrated that naturally-derived, biodegradable hybrid hydrogel scaffolds fabricated from a 10:1 w/w combination of alginate and type I collagen allow for maximal human umbilical vein endothelial cell (HUVEC) adherence and invasion in an in vitro wound healing model. We next sought to stimulate endothelial tubule formation within these scaffolds. METHODS: Hybrid hydrogel scaffolds were fabricated as before. Scaffolds were seeded with 3.0×105 HUVECs and maintained under standard cell culture conditions. After 3 days, culture media was supplemented with basic fibroblast growth factor (bFGF) at varying physiologic concentrations. Additional scaffolds were seeded with human aortic smooth muscle cells (HASMCs), followed by HUVECs 3 days later. After 2 weeks of HUVEC culture, all scaffolds were labeled with DiI-Ac-LDL and DAPI, and imaged using confocal microscopy. RESULTS: HUVECs were maximally confluent on hybrid scaffolds when cultured with media supplemented with bFGF 20ng/mL (372.5±37.4) compared with 0ng/mL (250.7±18.2, p=0.005), 1ng/mL (179.0±17.4, p<1.0x10-2), 5ng/mL (198.5±17.2, p<1.0x10-3), and 10ng/mL (273.3±22.9, p=0.030). However, the addition of growth factor stimulated neither an increase in HUVEC invasion nor tubule formation within the scaffold substrate. In contrast, a “dose-dependent” increase in tubule content and network-like organization was observed as the co-culture ratio of HASMCs to HUVECS from increased from 1:10 to 1:1 (Figures 1-2).Figure 1: Co-culture of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) in a 1:10 ratio within hybrid hydrogel scaffolds resulted in clusters of cells without an overlying structure or pattern. Scale bar = 100um.Figure 2: Co-culture of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) in a 1:1 ratio within hybrid hydrogel scaffolds resulted in an increase in tubule formation and network organization. Scale bar = 100um.CONCLUSION: Even at high concentrations, the presence of bFGF alone is insufficient to stimulate HUVECs to form tubules within hydrogel scaffolds. Instead, the complex chemical and possibly contextual cues offered by vascular smooth muscle cells are required to instruct endothelial cells to assemble into vasculogenic configurations. These findings provide important insights into the instruction of endothelial tubule and network formation and bring us one step closer to the fabrication of pre-vascularized artificial tissue. DISCLOSURES: This work was supported in part by the Ruth L. Kirschstein National Research Service Award (T32 HL083824-05) Institutional Research Training Grant (Dr. Reiffel), and the Weill Cornell Medical College Clinical and Translational Science Center (TL1RR024998; Dr. Fullerton).

Influence of alginate-based edible coating as carrier of antibrowning agents on bioactive compounds and antioxidant activity in fresh-cut Kent mangoes

The effect of alginate-based edible coating as carrier of antibrowning agents (ascorbic and citric acid) on color, bioactive compounds content and antioxidant activity of fresh-cut mangoes ‘Kent’ stored at 4 °C were evaluated. The application of alginate edible coating in conjunction with antibrowning agents (ascorbic and citric acid) to mango cubes maintained higher color values (L* and °Hue) and increased vitamin C content compared to mango cubes treated only with alginate coating or control. Total phenols content, measured throughout the Folin–Ciocalteu method, was increased 2-fold in cubes coated with alginate plus antibrowning agent. This behavior was attributed to vitamin C addition and not to a real increase in phenolic compounds. However, mangoes coated with alginate plus antioxidant presented higher concentration of ρ-hydroxybenzoic and ellagic acid than those samples coated just only with alginate or control. Mango cubes with antibrowning agents had the highest antioxidant activity due the ascorbic acid addition. The combination of alginate and antibrowning agent preserved the color of fresh-cut mangoes and increased the antioxidant potential of cubes.

Sustained-release alginate-chitosan pellets prepared by melt pelletization technique

Context: Alginate-chitosan pellets prepared by extrusion-spheronization technique exhibited fast drug dissolution.Objective: This study aimed to design sustained-release alginate pellets through rapid in situ matrix coacervation by chitosan during dissolution.Methods: Pellets made of alginate with chitosan and/or calcium acetate were prepared using solvent-free melt pelletization technique which prevented reaction between processing materials during agglomeration and allowed such reaction to occur only in dissolution phase.Results: Drug release was retarded in pH 2.2 medium when pellets were formulated with calcium acetate or chitosan till a change in medium pH to 6.8. The sustained-release characteristics of calcium alginate pellets were attributed to pellet dispersion and rapid cross-linking by soluble Ca2+ during dissolution. The slow drug release characteristics of alginate-chitosan pellets were attributed to polyelectrolyte complexation and pellet aggregation into swollen structures with reduced erosion. The drug release was, however, not retarded when both calcium acetate and chitosan coexisted in the same matrix as a result of chitosan shielding of Ca2+ to initiate alginate cross-linkages and rapid in situ solvation of calcium acetate induced fast pellet dispersion and chitosan losses from matrix.Conclusion: Similar to calcium alginate pellets, alginate-chitosan pellets demonstrated sustained drug release property though via different mechanisms. Combination of alginate, chitosan and calcium acetate in the same matrix nevertheless failed to retard drug release via complementary drug release pattern.

In Vivo study of a blended hydrogel composed of pluronic F-127-alginate-hyaluronic acid for its cell injection application

The epitome that cell carrier serves solely as passive vehicles has become outdated. It is now evident that the carrier microenvironment also contributes in the regeneration process. In this study, a combination of alginate, pluronic F127 and extracellular matrix (ECM) component, hyaluronic acid (HA) based scaffold has been prepared for in situ gelling vehicles for muscle cells. ECM incorporated blended hydrogel showed enhanced uniform distribution of muscle cells in a nude mouse model forming the scaffold in situ allowed the muscle cells to proliferate efficiently, indicating that a pluronic F127/alginate/HA matrix provided a beneficial environment for cellular growth and expansion. The formation of gel beneath the skin of nude mice was confirmed using optical coherence tomography (OCT). OCT has been used to visualize the in situ localization of cells as well. This in situ gelation is found to be advantageous for regenerative applications due to the absence of toxic solvents or co-polymerization agents; besides the handling process is simple. This study demonstrates that an in situ blended hydrogels enables the favorable settlement of cells and satisfactory cell delivery for muscle regeneration applications.

121 ALGINATE-FIBRIN GEL MATRIX PROMOTES IN VITRO GROWTH OF DOG SECONDARY FOLLICLES

A previous study from our laboratory has demonstrated that preantral follicles from the dog that are cultured in alginate are able to grow and produce steroid hormones (Songsasen et al. 2011 Reproduction 142, 113–122). Here we investigated the influence of using a combination of alginate and a degradable biomaterial, fibrin, on dog follicle development in vitro. We hypothesised that the alginate and fibrin gel matrix would be superior to alginate alone because the former has dynamic mechanical properties that permit more expansive follicle development than the inert alginate-only system. Secondary follicles (128–220 μm in diameter) were collected from the ovaries of 4 prepubertal dogs (&lt;6 months of age) and encapsulated in 0.5% alginate (n = 26) or 0.5% alginate + 12.5 mg mL–1 of fibrin (n = 22). Follicles were cultured for 12 days at 38.5°C in 100 μL of α-minimal essential medium + 2 mM of glutamine + 5.5 μg mL–1 of insulin + 5.5 μg mL–1 of transferrin + 6.7 ng mL–1 of selenium + 10 μg mL–1 of FSH and 1 ng mL–1 of LH + 3 mg mL–1 of polyvinyl alcohol. Follicle diameter was monitored and half of the medium exchanged every 48 h. Follicle survival was assessed based on ability to increase in size, as well as on oocyte and granulosa cell morphology. Comparisons of follicle growth rate for each culture day between the 2 treatments were conducted using Student's t-test and among culture days within the same group using ANOVA followed by a Holm-Sidak multiple comparison. Follicle survival was compared using a chi-square test. In both groups, follicles maintained the 3-dimensional structure and increased (P &lt; 0.05) in size as culture period progressed. However, follicles encapsulated in alginate + fibrin grew larger (P &lt; 0.05) than those in alginate alone. Specifically, follicles in alginate + fibrin were doubled in size by 12 days compared with a 60% increase for alginate alone. There were no differences (P &gt; 0.05) in follicle survival between the 2 groups (27.0 and 38.1% for alginate and alginate-fibrin, respectively). Results demonstrate that a dynamic alginate-fibrin matrix enhances in vitro follicle growth. We suspect that the mechanism involved is related to facilitating expansion capacity. Specifically, it is likely that nondegradable alginate offers physical, but eventually restrictive, support to encapsulated cells. By contrast, in the gel combination, the fibrin degrades due to cell-secreted proteases that, in turn, permit more robust follicle expansion. Low follicle survival (&lt;40%) in both treatments emphasises the need for more studies to identify influential endocrine/paracrine factors that enhance follicle growth and production of competent oocytes. Funded by NIH-KO1RR020564.

Recycling waste debris of immobilized microalgae and plant growth-promoting bacteria from wastewater treatment as a resource to improve fertility of eroded desert soil

This study attempted to demonstrate that biological residue from a new biological wastewater treatment is a resource for improving quality of arid soils and plant growth. After tertiary wastewater treatment, debris composed of alginate beads containing the microalgae Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense was used as an amendment for eroded, infertile desert soil having low levels of organic matter. A. brasilense survived in these used dried beads for at least one year. Three consecutive applications of the dry debris increased organic matter, organic carbon, and microbial carbon in the soil. Growth of sorghum in the amended soil was greater than plants grown in low organic matter, untreated soil or soil amended with beads containing other combinations of alginate, microalgae, or bacteria. The surface of plant roots growing in the amended soil was heavily colonized by A. brasilense, with no endophytic colonization; root tips were the preferred sites of colonization.

Storage Stability of Phenolic-Fortified Avocado Oil Encapsulated Using Different Polymer Formulations and Co-extrusion Technology

This study compares the stability and phenolic contents of avocado oil fortified with phloridzin (at 300 ppm) and encapsulated with alginate or the combination of alginate and hydroxypropyl methylcellulose (HPMC) (at a ratio of 3:1), over a 90-day storage period at 37°C. The storage stability of unencapsulated avocado oil (control), and encapsulated oils without any added antioxidant, or with butylated hydroxytoluene (BHT) were also examined for comparison. Results of peroxide value, p-anisidine value, Totox value, free fatty acid and extractable phenolic content analyses suggest that the combined use of encapsulation and an antioxidant (BHT or phloridzin) were synergistically beneficial for improving the oxidative stability and suppressing hydrolytic rancidity of avocado oil at 37°C. Alginate alone was a better encapsulant for avocado oil than the combination of alginate and HPMC, which was supported by the results of optical and environmental scanning electron microscopy examinations which showed that alginate beads were bigger and stronger than alginate–HPMC beads. The finding that phloridzin was still significantly retained in the encapsulated oil after the 90-day storage indicated that the final encapsulated oil product possessed additional nutritional values derived from this phenolic antioxidant. Thus, both the encapsulant polymer matrix and the types of added antioxidant are ultimately important when using the antioxidant fortification and oil encapsulation approaches for tackling oil stability issues.

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA-alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 degrees C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)-alginate beads with three different sizes (1-3 mm), at three different alginate concentrations (0.2-1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 degrees C for the PVA-alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)-alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 degrees C. The Michaelis constant (K(Mapp)) and the maximum reaction velocity (V(maxapp)) were evaluated for both free (K(Mapp) = 0.233 mM; V(maxapp) = 0.13 mM min(-1)) and immobilized naringinase (K(Mapp) = 0.349 mM; V(maxapp) = 0.08 mM min(-1)). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 degrees C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.

Quality and acceptability of a set-type yogurt made from camel milk

Camel milk (CM) set yogurts were formulated with gelatin, alginate (ALG), and calcium (Ca). Titratable acidity, pH, sensory properties, and acceptability of CM yogurts were studied. Twelve treatments were prepared; 3 using gelatin at 0.5, 0.75, and 1% levels and 9 with combinations of ALG and Ca at different levels. Titratable acidity and pH of fresh yogurt were not affected by the addition of gelatin or the ALG and Ca combinations. Trained sensory panel results showed that CM yogurt containing 1% gelatin or 0.75% ALG + 0.075% Ca had the highest intensities for firmness and body. Consumer results indicated that the hedonic ratings of the sensory attributes and acceptability of CM yogurt containing 0.75% ALG + 0.075% Ca were similar to that of cow's milk yogurt. The CM yogurts containing ALG + Ca and flavored with 4 different fruit concentrates (15%) had similar hedonic ratings and acceptability. Addition of 0.75% ALG + 0.075% Ca could be used to produce acceptable plain or flavored CM yogurt.

Cell-Laden and Cell-Free Biopolymer Hydrogel for the Treatment of Osteochondral Defects in a Sheep Model

The objective of the current study was to determine the suitability of cell-laden and cell-free alginate-gelatin biopolymer hydrogel for osteochondral restoration in a sheep model (n = 12). Four femoral defects per animal were filled with hydrogel (cHG) plus autologous chondrocytes (cHG + C) or periosteal cells (cHG + P) or gel only (cHG) or were left untreated (E). In situ solidification enabled instantaneous implant fixation. Sixteen weeks postoperatively, defect sites were processed for light microscopy and immunofluorescence. A modified Mankin and a semi-quantitative immunoreactivity score were used to evaluate histology and immunofluorescence, respectively. Defects after cHG + C were restored with smooth, hyaline-like neo-cartilage and trabecular subchondral bone. cHG + P and cHG treatments revealed slightly inferior regenerate morphology. Undifferentiated tissue was found in E. The histological score showed significant (p < 0.05) differences between all treatment groups. In conclusion, cHG induces satisfactory defect regeneration. Complete filling of the cavity in one step and subsequent rapid in situ solidification was feasible and facilitated graft fixation. Cell implantation might be beneficial, because cells seem to play a key role in histological outcome. Still, their contribution to the repair process remains unresolved because host cell influx takes place. The combination of alginate and gelatin, however, creates an environment capable of serving implanted and host cells for osteo-chondrogenic tissue regeneration.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture

The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

In vitro permeability of round window membrane to transforming dexamethasone with delivery vehicles — a dosage estimation

In recent years the interest of sustained drug delivery into inner ear is promising, at the same time a great deal of novel oral drugs using biodegradable vehicles have been produced to achieve sustained drug release. The aim of this study was to use biodegradable vehicles to release dexamethasone in the round window membrane application. Dexamethasone gels composed of alginate and chitin were prepared and the release-permeating profiles were studied using a reproducible in vitro apparatus. A longer-period time course was simulated using the parameters acquired in this study. The data obtained in this study was compared with those of other studies in intratympanic drug delivery, and an appropriate initial dosage was extrapolated. The combination of alginate and chitin could efficiently restrict dexamethasone diffusion and the time course suggested a sustained drug concentration within 24 hours. A higher initial dosage was estimated to achieve a stable therapeutic concentration in vivo. The combination of alginate and chitin could be used as vehicle for sustained release of dexamethasone in intratympanic application.

Technological aspects for restructuring concentrated pineapple pulp

This study investigated how different concentrations of alginate, low methoxy pectin and glycerol could be used to obtain restructured concentrated pineapple pulp, with the aid of response surface methodology. The results of a central composite design show that changes in pectin and alginate levels have statistically significant effects on the firmness of restructured pineapple fruit. Products with the highest values for firmness resulted from using 16 g/kg of both alginate and pectin independent of the concentration of glycerol used. Glycerol, used at the concentration of 100 g/kg, reduced the water activity of the restructured product to an intermediate moisture level (0.880), besides substituting part of the sucrose incorporated into the fruit pulp. A mixture of low methoxy pectin and alginate made it possible to obtain very firm structured fruits using acid pulps. The restructured fruit formulated with a high content of concentrated pineapple pulp (735 g/kg), low sugar content (210 g/kg) and a combination of alginate (20 g/kg), pectin (10 g/kg) and glycerol (100 g/kg), partially dehydrated to 20% moisture content (w.b.), was evaluated by a group of panelists and received a mean score of 5.45, between “liked slightly” and “liked moderately” on the seven-point hedonic scale.

Endoluminal hydrogel films made of alginate and polyethylene glycol: Physical characteristics and drug‐eluting properties

Drug-eluting stents signify a major achievement in reducing the incidence of coronary restenosis after percutaneous transluminal coronary angioplasty. However, where drug-eluting stents have been unsuccessful, endoluminal gel-paving strategies offer renewed optimism, mainly in a variety of vascular procedures requiring catheter-based sustained, localized delivery of therapeutic drugs, and biological factors. Despite promising results in animals, endoluminal paving has met with very limited clinical success because of the technical difficulties and stringent safety demands. The current study presents an alternative to gel paving using 40-mum-thick biodegradable polymeric films for deployment onto the artery wall during balloon angioplasty and stenting. The films are made from a durable yet compliant network of alginate and polyethylene glycol (PEG), and are securely held affixed to the vessel wall by the expanded stent struts. The alginate-based films are characterized by measuring their strength, elasticity, degree of swelling, degradability in water and saline, and drug release properties. The combination of alginate and PEG afforded the films sufficient strength and compliance for endoluminal deployment using an in vitro organ culture system. In characterizing the film degradability, it was discovered that the ionic concentration of the buffered saline was the main determinant in regulating the degradation kinetics and the release kinetics of the drug molecule Paclitaxel. These results suggest that the use of alginate-based, PEG-containing polymeric films for endoluminal coverage offers an alternative solution to conventional drug-eluting stents, with the added advantage of uniform endoluminal coverage of the treated segment and homogeneous endoluminal application of the active substance.

Alginate–pectin microcapsules as a potential for folic acid delivery in foods

Most naturally occurring folate derivatives in foods are highly sensitive to temperature, oxygen, light and their stability is affected by processing conditions. Folic acid incorporated microcapsules using alginate and combinations of alginate and pectin polymers were prepared to improve stability. Folic acid stability was evaluated with reference to encapsulation efficiency, gelling and hardening of capsules, capsular retention during drying and storage. Use of alginate in combination with pectin produced more robust capsules and contributed to greater encapsulation efficiency. The capsules lost their spherical shape as a consequence of increased pectin. The high alginate capsules, A100:P0 (100% alginate: 0% pectin) and A80:P20 (80% alginate: 20% pectin) were of regular spherical shape, while those with more pectin, A70:P30 (70% alginate: 30% pectin) and A60:P40 (60% alginate: 40% pectin) formed irregular spheres. The loading efficiency, expressed as a percentage of the actual loading to theoretical loading, varied from 55–89% with the composition of the mixed polymers. After 11 weeks of storage at 4°C, folic acid retention in freeze-dried capsules was 100% (A70:P30 and A60:P40), 80% (A80:P20) and 30% (A100:P0). The blended alginate and pectin polymer matrix increased folic acid encapsulation efficiency and reduced the leakage from the capsules compared to those made with alginate alone and showed higher folic acid retention after freeze drying and storage.

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension

Objective To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture.Methods Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O2for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [3H]-proline incorporation and cell-viability by the trypan blue exclusion method.Results The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [3H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [3H]-proline incorporation. Viability was ∼90% for all alginate cultures.Conclusion A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.

Comparison of three methods for culturing throat swabs from cystic fibrosis patients

In patients with cystic fibrosis who do not produce sputum, deep throat swabs are cultured for potential respiratory pathogens. Usually these swabs are directly streaked onto selective agar media. In a study of 50 pediatric cystic fibrosis patients, we compared this traditional method using rayon swabs with two methods having quantitative modifications: calcium alginate swabs eluted in Ringer's lactate and rayon swabs eluted in normal saline. The eluates were then processed quantitatively (three-step dilution series). The yield of potential pathogens was significantly higher with the two quantitative methods. Overall, the combination of alginate with Ringer's lactate was superior to the combination of rayon with saline, although only some of these differences achieved statistical significance.