Laboratory Investigations Dentinal Proteoglycans Demonstrate an Increasing Order of Affinity for Hydroxyapatite Crystals During the Transition of Predentine to Dentine A. M. Milan, 1 R. V. Sugars, 2 G. Embery, 1 R. J. Waddington 3 1 Department of Clinical Dental Sciences, The University of Liverpool, Liverpool, L69 3GN, United Kingdom 2 Centre for Oral Biology, Karolinska Institutet, Stockholm, Sweden 3 Department of Dental Health and Biological Sciences, University of Wales College of Medicine, Cardiff, CF14 4XY, United Kingdom Received: 5 November 2003 / Accepted: 17 February 2004 / Online publication: 16 June 2004 Abstract. The transition from an unmineralized pred- entine to a mineralized dentine involves a variety of molecular extracellular matrix interactions and protein degradation events. Previous studies have identified that different pools of proteoglycan (PG) species are present within the matrix of the predentine, the transitional phase at the predentine–dentine border, and the miner- alized dentine. These PGs alter with respect to the chemical nature of the glycosaminoglycan (GAG) chain and as a result of extracellular processing of the mac- romolecule in the matrix. This study has examined the hydroxyapatite (HAP) binding affinity of the PGs iso- lated from these phases and the influence of the attached GAG chains upon their binding characteristics. PGs isolated from the three phases were characterized to contain a mixture of decorin and biglycan, substituted with chondroitin sulfate GAG chain(s). Maximal bind- ing for dentine PGs onto HAP was achieved at 15.60 lg/ ml protein and for predentine–dentine interface PGs at 0.125 mg/ml. A significantly increasing gradient of affinity was observed moving toward dentine, with dentine PGs exhibiting 19 times greater binding affinity for HAP than predentine PGs and 7.5 times greater affinity than predentine–dentine interface PGs. Removal of the GAG chains from dentine PGs significantly re- duced binding affinity for HAP but did not influence the number of binding sites. The difference in binding ability observed for the different PG pools gives further sup- port for the involvement of these macromolecules in regulating the transition from predentine to dentine and suggests key roles for the GAG chains in the minerali- sation process. Key words: Proteoglycans — Dentine — Mineralization — Hydroxyapatite Introduction Dentine formation and mineralization involves a dy- namic transition from an unmineralized predentine to a mineralized dentine. The process of dentinogenesis ap- pears to be controlled by the temporal expression of a range of extracellular matrix (ECM) proteins, which participate in a variety of molecular interactions and potentially undergo modification or processing within the extracellular environment of the predentine matrix (reviewed by [1]). Dentinogenesis therefore presents a model for biological mineralization, where the preden- tine, the predentine/dentine interface, and the mineral- ized matrix of dentine are ultrastructurally and biochemically discernible tissues. The molecular inter- actions and metabolic modifications of the ECM pro- teins present within these phases must clearly be directed in a logical and specific manner to enable dentinogenesis to proceed in an ordered and reproducible fashion. However, during various pathological conditions, including fluorosis, dentinogenesis imperfecta II, and osteoporosis, these mineralization patterns are disrupted. The present study has focused on the dentinal pro- teoglycans (PG), which are key ECM macromolecules involved in the mineralization processes of dentine and bone. Biochemical, histochemical, and immunohisto- chemical studies on the PGs of dentine and predentine have yielded sufficient information to indicate that PGs belonging to the small leucine-rich interstitial family (SLRPs) predominate [2–8]. This family consists of structurally related but genetically distinct macromole- cules, which include decorin, biglycan, lumican, fibro- modulin, keratocan, PRELP, osteoadherin, epiphycan, and osteoglycin, which are present in a wide variety of connective tissues [9]. The dentinal SLRPs have been identified as being predominantly chondroitin sulfate rich, with decorin and biglycan the major constituents Correspondence to: A. M. Milan; E-mail: ammilan@liv.ac.uk Calcif Tissue Int (2004) 75:197–204 DOI: 10.1007/s00223-004-0273-z