Effects of negatively charged groups (carboxymethyl) on the calcification of poly(2-hydroxyethyl methacrylate)

Poly(2-hydroxyethyl methacrylate) (pHEMA) has potentially wide biomedical applications: it is biocompatible, allows immobilization of cells or bioactive molecules and has a hardness comparable to bone. We previously reported that immobilization of alkaline phosphatase (AlkP) in pHEMA can initiate mineralization in a manner that mimics the calcification of cartilage and woven bone. Because numerous proteins known to initiate mineralization possess acidic species, we have modified the neutral electrical surface of pHEMA by carboxymethylation (CM). We have studied the effects of these negative groups on the calcification process in vitro. Calibrated pellets of pHEMA were prepared and carboxymethylated by soaking with 0.5 M bromoacetic acid in 2 M NaOH. Pellets of pHEMA, pHEMA-AlkP and pHEMA-CM were incubated during 5, 10 and 15 days in two types of body fluid: normal (1X) and 1.5X concentration of ions. Nodules of hydroxyapatite developed on pHEMA-AlkP and pHEMA-CM but not on pHEMA. Hydroxyapatite crystals were dissolved in HCl allowing calcium to be dosed. CM significantly increased the amount of deposited Ca by 1.8 folds in the 1X fluid and 15.8 folds in the 1.5X fluid. The presence of AlkP considerably increased the amount of deposited Ca: 25.9 folds in 1X and 23.3 in 1.5X. ROS 17/2.8 osteoblast-like cells were seeded on the materials and examined by confocal microscopy after phalloidin staining. Cells grown on pHEMA alone appeared round, while cells grown on the crystals deposited on the pHEMA-CM or pHEMA-AlkP were flattened. The presence of AlkP favours the mineralization process more than the existence of surface negative groups on the polymer. Cells preferentially adhere to the polymer when hydroxyapatite crystals were developed.