Abstract
Abstract: :
Purpose: the main goal of this study is to evaluate the effects of matrix composition on the development of endothelial cells (Ec) capillary sprouts with particular attention on the role played by collagen and hyaluronic acid (HA) on a 3D model of angiogenesis. Materials and Methods: two types of Ec cells are used for our study: Human Umbilical Vein Endothelial (HUVE) cells, isolated from human umbilical veins of newborn babies using collagenase digestion, and Pig Aortic Endothelial (PAE) cells cultured at 37° C (5% CO2 , 100% humidity) in HAM’s F12 Media with 10% fetal bovine serum and 1% pen–strep. Collagen was used in a 1.0 mg/mL concentration mixed with either medium or HA to a final concentration of 5.0 mg/mL. Approximately 300 µL of the material was pipetted into 8 well chamber slides. After an hour in the incubator, the collagen was fully fibrillated and the appropriate medium is placed in each incubator. Cells are studied with a confocal microscope directly or with a fluorescent labeling. Results:the matrix composition, collagen or a mixture of collagen and hyaluronic acid, has the greatest effect on angiogenesis. After 3 hours, both materials have similar influences on the sprouting. After 18 hours, the effect is quite evident and the spheroids in the HA and collagen mix have still not sprouted. After 18 hours in just collagen, angiogenesis is unhampered and sprouting is prominent. The HA does not seem to have a negative affect on the overall sprouting, at long times, the spheroids in HA/collagen mix have just as much sprouting as in the collagen–only wells. Conclusions:The delay in sprouting coincides with a respective delay in the formation of stress fibers between spheroids. While spheroids embedding in collagen matrices form stress fibers within 3 hours, spheroids in a collagen and HA matrix take up to 48 hours to form similar stress fibers. The HA addition to collagen produces a physical change in the matrix as seen from SEM photos, but does not alter the mechanical properties. The soluble factor molecules are small enough to freely diffuse throughout both matrices with collagen and collagen and HA with the same ease, they should be able to reach the spheroids in similar times in both materials. Since we observe a delay in stress fibers, the material aspects have an effect on angiogenesis. The two possibilities to explain this delay is that the addition of HA changes the collagen structure, making it more difficult to remodel the fibers, or that the HA coating on the collagen blocks the spheroids from the receptors on the collagen matrix, delaying the migration.
Keywords: growth factors/growth factor receptors • imaging/image analysis: non–clinical • motion–3D