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Richard Yi-Jen Hwang, Raul Velez, Sergio Groman-Lupa, Yu Cheol Kim, Jeffrey Olson; Adsorption of VEGF to Hydroxyapatite. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):149. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Diseases such as exudative macular degeneration and diabetic retinopathy are characterized by increased levels of VEGF. Current therapies to curtail the effects of VEGF necessitate the use of repeated intravitreal injections. This study tests the hypothesis that a solid implant of hydroxyapatite could be used to trap and denature VEGF.
Adsorption experiments were performed using diluted recombinant human VEGF. Three groups (each group n=3) were tested: hydroxyapatite (300g dry weight) , acrylic (sham control), and VEGF alone (negative control). Each was placed in a container with a VEGF concentration of 400 pg/ml. Each was then incubated at 4 degrees C for 24 hours and final free concentrations of VEGF were measured via ELISA. Total VEGF adsorbed was then calculated by subtracting the final and initial concentrations.<br /> <br /> Denaturing experiments were performed using confocal microscopy. Hydroxyapatite blocks incubated with fluorescently labeled VEGF overnight were surface ablated with 532 nm laser and re-imaged to evaluate focal decreases in fluorescence. Laser treated hydroxyapatite was then re-incubated with VEGF.
Hydroxyapatite significantly reduced VEGF concentration in vitro when compared to acrylic and controls. VEGF concentrations with HA were 64 +/- 3 pg/ml (p < 0.001 vs acrylic and control) versus 358 +/- 6 and 368 +/- 5 pg/ml (p = 0.29 acrylic vs control) for acrylic and control groups. Hydroxyapatite and acrylic incubated with VEGF when rinsed with clean solution eluted only small levels of VEGF: HA 29 +/- 2pg/ml and acrylic 33 +/- 1 pg/ml (p=0.15). Student t-test was used to determine p values.<br /> <br /> Application of ablative laser to hydroxyapatite containing adsorbed fluorescent VEGF decreased local surface fluorescence. These areas treated with laser can then re-adsorb additional VEGF.
These results are consistent with the hypothesis that solid hydroxyapatite can be used to trap VEGF in vitro. By applying laser to the hydroxyapatite surface, bound proteins can be denatured. Further testing is needed, but this concept could prove useful in treating diseases characterized by high intraocular levels of VEGF.
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