BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

UNLABELLED CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria

The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and poly-L-lysine in combination with alginate. The thickness of the alginate beads increased with the addition of coating materials. No differences were detectable in the bead strength by texture analysis or in the thickness of the beads with different types of coating materials by transmission electron microscopy. The survivability of three probiotics in uncoated beads, coated beads, and as free cells (unencapsulated) was conducted in 0.6% bile salt solution and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated heads. (C) 2004 Elsevier Ltd. All rights reserved.

Nanoassembly of biocompatible microcapsules for urease encapsulation and their use as biomimetic reactors

Biocompatible polypeptide capsules with high enzyme loading and activity prepared by templating mesoporous silica spheres were used as biomimetic reactors for performing CaCO3 synthesis exclusively inside the capsule interior via urease-catalyzed urea hydrolysis.

Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage

Survival of the microencapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, in stirred yoghurt from UHT- and conventionally treated milk during low temperature storage was investigated. The probiotic cells both as free cells and microencapsulated cells (in alginate beads coated with chitosan) were added into 20 g/100 g total solids stirred yoghurt from UHT-treated milk and 16 g/100 g total solids yoghurt from conventionally treated milk after 3.5 h of fermentation. The products were kept at 4 degrees C for 4 weeks. The survival of encapsulated probiotic bacteria was higher than free cells by approximately 1 log cycle. The number of probiotic bacteria was maintained above the recommended therapeutic minimum (10(7) cfu g(-1)) throughout the storage except for R bifidum. The viabilities of probiotic bacteria in yoghurts from both UHT- and conventionally treated milks were not significantly (P > 0.05) different. (c) 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-(n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm2) caused the release of liposome-entrapped CaCl2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production. © 2007 Elsevier B.V. All rights reserved.