Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes.

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.

Films of agarose enable rapid formation of giant liposomes in solutions of physiologic ionic strength.

This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield. This method did not require the presence of an electric field or specialized lipids; it generated giant liposomes from pure phosphatidylcholine lipids or from lipid mixtures that contained cholesterol or negatively charged lipids. Hybrid films of agarose and lipids even enabled the formation of giant liposomes in PBS from lipid compositions that are typically problematic for liposome formation, such as pure phosphatidylserine, pure phosphatidylglycerol, and asolectin. This paper discusses biophysical aspects of the formation of giant liposomes from hybrid films of agarose and lipids in comparison to established methods and shows that gentle hydration of hybrid films of agarose and lipids is a simple, rapid, and reproducible procedure to generate giant liposomes of various lipid compositions in solutions of physiologic ionic strength without the need for specialized equipment.

VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects.

UNLABELLED: Bone regeneration is challenging in sites where the blood supply has been compromised by radiation. We examined the potential of a growth factor (VEGF) delivery system to enhance angiogenesis and bone formation in irradiated calvarial defects. VEGF-releasing polymers significantly increased blood vessel density and vascular perfusion in irradiated defects and increased bone formation relative to control conditions. INTRODUCTION: Radiation therapy causes damage to tissues and inhibits its regenerative capacity. Tissue injury from radiation is in large part caused by a compromised vascular supply and reduced perfusion of tissues. The aim of this study was to determine if delivery of vascular endothelial growth factor (VEGF) from a biodegradable PLGA (copolymer of D,L-lactide and glycolide) scaffold could enhance neovascularization and bone regeneration in irradiated osseous defects. MATERIALS AND METHODS: An isolated area of the calvarium of Fisher rats was irradiated (12 Gy) 2 weeks preoperatively, and two 3.5-mm osseous defects were created in this area, followed by the placement of PLGA scaffolds or VEGF scaffolds (PLGA scaffolds with incorporated VEGF) into the defects. Laser Doppler perfusion imaging was performed to measure perfusion of these areas at 1, 2, and 6 weeks. Implants were retrieved at 2, 6, and 12 weeks, and histologic and muCT analyses were performed to determine neovascularization and bone regeneration. RESULTS: Histological analyses revealed statistically significant increases in blood vessel formation (>2-fold) and function (30%) within the VEGF scaffolds compared with PLGA scaffolds. Additionally, evaluation of bone regeneration through bone histomorphometric and muCT analyses revealed significantly greater bone coverage (26.36 +/- 6.91% versus 7.05 +/- 2.09% [SD]) and increased BMD (130.80 +/- 58.05 versus 71.28 +/- 42.94 mg/cm(3)) in VEGF scaffolds compared with PLGA scaffolds. CONCLUSIONS: Our findings show that VEGF scaffolds have the ability to enhance neovascularization and bone regeneration in irradiated osseous defects, outlining a novel approach for engineering tissues in hypovascular environments.

Endothelial cell modulation of bone marrow stromal cell osteogenic potential.

In the context of bone development and regeneration, the intimate association of the vascular endothelium with osteogenic cells suggests that endothelial cells (ECs) may directly regulate the differentiation of osteoprogenitor cells. To investigate this question, bone marrow stromal cells (BMSCs) were cultured: in the presence of EC-conditioned medium, on EC extracellular matrix, and in EC cocultures with and without cell contact. RNA and protein were isolated from ECs and analyzed by reverse transcriptase-polymerase chain reaction and Western blotting, respectively, for expression of bone morphogenetic protein 2 (BMP-2). In animal studies, BMSCs and ECs were cotransplanted into severe combined immunodeficient mice on biodegradable polymer matrices, and histomorphometric analysis was performed to determine the extent of new bone and blood vessel formation. ECs significantly increased BMSC osteogenic differentiation in vitro only when cultured in direct contact. ECs expressed BMP-2, and experiments employing interfering RNA inhibition confirmed its production as contributing to the increased BMSC osteogenic differentiation. In vivo, cotransplantation of ECs with BMSCs resulted in greater bone formation than did transplantation of BMSCs alone. These data suggest that ECs function not only to form the microvasculature that delivers nutrients to developing bone but also to modulate the differentiation of osteoprogenitor cells in vitro and in vivo.