Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature.

Injectable scaffolds are of interest in the field of regenerative medicine because of their minimally invasive mode of delivery. For tissue repair applications, it is essential that such scaffolds have the mechanical properties, porosity and pore diameter to support the formation of new tissue. In the current study, porous poly(dl-lactic acid-co-glycolic acid) (PLGA) microspheres were fabricated with an average size of 84±24µm for use as injectable cell carriers. Treatment with ethanolic sodium hydroxide for 2min was observed to increase surface porosity without causing the microsphere structure to disintegrate. This surface treatment also enabled the microspheres to fuse together at 37°C to form scaffold structures. The average compressive strength of the scaffolds after 24h at 37°C was 0.9±0.1MPa, and the average Young's modulus was 9.4±1.2MPa. Scaffold porosity levels were 81.6% on average, with a mean pore diameter of 54±38µm. This study demonstrates a method for fabricating porous PLGA microspheres that form solid porous scaffolds at body temperature, creating an injectable system capable of supporting NIH-3T3 cell attachment and proliferation in vitro.

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

Cell-type-specific adhesion onto polymer surfaces from mixed cell populations.

The targeted adhesion of a specific cell type from a mixed cell suspension via the surface presentation of a cell-specific ligand is demonstrated. This generic strategy is illustrated by the covalent attachment of a galactose derivative to a polylysine backbone via the amine functionality. Following adsorption of the resultant material to a polymer surface, hepatocyte adhesion is increased via the interaction between galactose and asialoglycoprotein receptors in a concentration-dependent manner. The selective nature of the material is demonstrated by the approximate doubling in the adhesion of hepatocytes relative to a nontargeted cell type (hepatic stellate cells), and an inability of the modified polymer surface to attract additional numbers of the nontargeted cells. This strategy provides a mechanism for controlling the ratios of cell types adhering to scaffold supports, thus enabling the rapid creation of defined coculture systems from heterogeneous cell suspensions.