Diffusion Systems for Evaluation of Biomineralization

Abstract

A variety of in vitro study methods have been used to elucidate the roles of matrix molecules in biomineralization processes. Among these, gel diffusion-precipitation studies have proved to be an effective tool. This methodology is uniquely capable of characterizing the effects of matrix molecules on mineralization while only using very small quantities of material. Furthermore, gel methods have been extended for use as a mineralization assay system to characterize modified matrix molecules and synthetic analogues. Here we discuss the advantages and limitations of gelatin, agar, agarose, and other systems for studying the mechanisms of biomineralization. KeywordsHydroxyapatite; Biomineralization; Gel diffusion; Gelatin; Agarose; Agar In Vitro StudiesThe functions of vertebrate and non-vertebrate mineralized tissue matrix biomolecules, such as osteopontin [1][2][3][4], osteonectin [5,6], amelogenin [7,8], bone sialoprotein [2,3,6,9], and a variety of shell proteins [10,11] generally have been demonstrated in a three-step process. First, molecules that may influence the biomineralization process are either isolated from mineralized tissue, purified, and characterized, or cloned and expressed in recombinant forms. Their spatial and temporal distribution in mineralizing tissue, and their presence or absence from diseased or non-mineralizing tissue, as assessed by immunolocalization or in situ hybridization, suggests their role in mineral formation. Second, the molecule being studied can be associated with the biomineralization process in vivo via tissue studies on genetically modified or diseased organisms where expression of the substance of interest is altered, prevented, or enhanced. Third, in vitro mineralization studies may be used to provide insight into the mechanism of action of matrix proteins and to elucidate a molecule's function in vivo [12]. While many different techniques have been used in the in vitro studies, gel precipitation methods, used for over a century for the growth of large crystals [13], have recently been introduced to help elucidate how non-collagenous proteins and other biomolecules act to control biomineralization processes.The interplay of in vivo and in vitro studies allows characterization of a molecule's role in complex living tissue, where we have limited experimental control, against a backdrop of information from well-controlled, simpler precipitation studies. The cell-free in vitro studies allow characterization of an individual compound or mixture's effects on nucleation, growth kinetics, inhibition, and crystal composition and habit, as well as quantitative comparisons We review here the recent application of crystal formation in gels to probe mineralization mechanisms with emphasis placed on hydroxyapatite formation and growth, although the techniques described are applicable to other types of biomineralization.Most in vitro studies of matrix proteins involved in hydroxyapatite (HA) formation have been done in solution, as opposed to gels. The solution studies charact...

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