Modeling of matrix vesicle biomineralization using large unilamellar vesicles

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.