Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: effects of seeding density.

Autologous cell transplantation is a promising approach for cartilage repair, but the expansion of chondrocytes in a monolayer, a common approach to amplifying the cell number, inevitably leads to cell de-differentiation. To explore whether porous alginate sponges could be utilized for chondrocyte expansion and investigate the effects of seeding densities, the porcine chondrocytes were seeded to porous alginate sponges at low (5 x 10(5) cells per 40 sponges), medium (5 x 10(6) cells per 40 sponges), or high (2 x 10(7) cells per 40 sponges) density. After 4-week perfusion culture, all three groups resulted in chondrocyte proliferation, maintenance of chondrocytic gene (collagen II, Sox 9 and aggrecan) expression, and formation of cell clusters resembling cartilaginous tissues. The higher the seeding density, the higher the final cell density and GAGs production and, accordingly, the larger the cell clusters. Strikingly, the cumulative expansion ratios achieved by the low-density group ( approximately 150-fold) significantly exceeded those achieved by the medium (approximately 21-fold) and high (approximately 4.7-fold) density groups, as well as those achieved using other scaffolds. In conclusion, seeding chondrocytes to the alginate sponges at a low density, combined with perfusion culture, represents a drastic improvement in expanding autologous chondrocytes.

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli.

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either gamma-GCS or GS and found that gamma-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2-5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was approximately 2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli-based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents.