Figure 2.

TGFβ1 is bound and released by µ-beads. (A) Modification of porous agarose µ-beads with GAG is achieved by reaction in presence of EDC, enabling formation of covalent bonds between GAG’s acidic groups and amine groups of µ-beads. The GAG provides binding sites for TGFβ1 adsorption. TGFβ1 adsorption is concentration dependent. Release is driven by a concentration difference between µ-beads’ interior and outside. (B) The GAG used in this study was medium-sulfated hyaluronan (msHA). The degree of sulfation is about 2 sulfate groups per disaccharide unit of the GAG and therefore similar to heparin. Possible positions of sulfate groups are marked with red ‘R’. (C) Release kinetics of TGFβ1 as determined by ELISA. Concentration of TGFβ1 was measured in the supernatant released from 10⁴ µ-beads. The depicted release curve is already re-calculated for cell culture conditions. It shows concentration increase of TGFβ1 in the supernatant delivered by 500 µ-beads. Released concentrations correlate with loading concentrations. The area shaded in grey (first 24 h) covers so-called “initial burst” of TGFβ1. This amount is washed out before cell culture experiments by repeated rinsing of collagen networks before seeding fibroblasts. (D) Increase of TGFβ1 concentration in the medium during cell experiments starting after cell seeding. The released concentration of TGFβ1 release over 24 h was subtracted. Release curves show TGFβ1 delivered by 500 µ-beads over 3 d from day 1 until day 4.