In vitro study of the effect of bisphosphonates on mineralization induced by a composite material: poly 2 (hydroxyethyl) methacrylate coupled with alkaline phosphatase
Filmon, R.; Baslé, M.F.; Barbier, A.; Chappard, D.
Morphologie Bulletin de l'Association des Anatomistes 84(264): 23-33
2000
ISSN/ISBN: 1286-0115 PMID: 10923337 Document Number: 518828
We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer (poly 2(hydroxyéthyl) methacrylate) (pHEMA) in a copolymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its biological 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 distribution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated with a synthetic body fluid containing organic phosphates (beta-glycerophosphate). Mineral deposits with a rounded shape (calcospherites) were obtained in about 17 days. We have investigated the effects of three bisphosphonates (etidronate, alendronate and tiludronate) on this system. 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 alendronate appeared to inhibit the calcospherite deposition onto the pHEMA-AlkP material in a similar way. Both bisphosphonates possess three sites for mineral complexion. On the other hand, tiludronate having only two sites was associated with a reduced inhibitory effect on mineralization. When used in microgravity conditions, mineralization was impaired with etidronate and larger crystals were obtained with tiludronate. However, these effects were obtained in non-physiological conditions (a 20 degrees C temperature was used during the STS80 flight of the space shuttle). The pHEMA-AlkP material provides an interesting method to study the effects of pharmacological compounds and environmental factors on the bone and cartilage mineralization process.