Poly(2-hydroxy ethyl methacrylate)-alkaline phosphatase: A composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process

We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer: poly(2-hydroxy ethyl methacrylate) - (pHEMA)- in a co polymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its enzymic activity. The image analysis of sections using a tessellation method showed a lognormal distribution of the area of the tiles surrounding AlkP particles, thus confirming a homogeneous distri bution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated w ith a synthetic body fluid containing organic phosphates (beta -glycerophos phate). Mineral deposits with a rounded shape (calcospherites) were obtaine d in about 17 days. We have investigated the effects of three bisphosphonic pharmacological compounds (etidronate, alendronate and tiludronate) on thi s system which mimics the mineralization process of cartilage and woven bon e. Bisphosphonates at a concentration of 10(-2) M totally inhibited AlkP in solution at a concentration of 10(-4) mg/ml. Inhibition has been reported being due to the chelation of a metal cofactor (Zn2+). Etidronate and alend ronate appeared to similarly inhibit the calcospherite deposition onto the pHEMA-AlkP material. Both bisphosphonates possess three sites for the miner al complexion by Ca chemisorbtion. On the other hand, tiludronate having on ly two sites, was associated with a reduced inhibitory effect on mineraliza tion but larger crystals were obtained. The pHEMA-AlkP material contains an immobilized enzyme in a hydrogel and mimics the physiological conditions o f matrix vesicles entrapped within the cartilage (or bone) matrix. It provi des an interesting method to study the effects of pharmacological compounds on the mineralization process in bone and cartilage in a non cellular and protein-free model.