Acidic matrix macromolecules involved in regulation of biological crystal growth often contain aspartic acid-rich domains and covalently bound sulfated polysaccharides. We propose that sulfates and 13-sheet structured carboxylates cooperate in oriented calcite crystal nucleation. The sulfates concentrate calcium, creating the' supersaturation necessary for nucleation on the structured carboxylate domains. An artificial model, composed of sulfonated polystyrene surfaces and adsorbed 13-sheet poly(aspartate), demonstrates that the two components indeed act cooperatively with respect to two independent assays, both by induction of calcite nucleation off the (001) plane and by calcium association. Evidence is presented that a purified organic matrix acidic glycoprotein from mollusk shells may behave in vitro in a similar way.Crystal formation by organisms is commonly controlled by an array of extracellular proteins and polysaccharides. In skeletal mineralization these macromolecules generally form a mold or framework in which the crystals grow. The macromolecules on the surfaces of this "organic matrix" are most directly involved in regulating crystal nucleation and growth. (7). Under these conditions the calcium ions show a nearly ideal Donnan distribution (8), indicating that they are not bound on a one-to-one basis to sulfate groups. Such an "ionotropic effect" (9) was in fact proposed as a mechanism for inducing crystal nucleation in mollusk shell formation (6). We therefore hypothesized that in our in vitro experiments (3, 4) the sulfate groups of the polysaccharides, which are covalently bound to the protein backbone, concentrate calcium. This creates the local supersaturation necessary for nucleation of calcite on the structured carboxylate domains.To test this hypothesis we created an artificial model substrate for crystal growth composed of sulfonated polystyrene films (10) to which poly(aspartate), in the P-sheet conformation, is adsorbed. The separate and combined effects of the two elements of the system are tested. Two independent "assays" are used: induction of (001)-oriented calcite nucleation and calcium association. We show that indeed sulfonates and structured carboxylates act cooperatively with respect to both assays, and we present evidence suggesting that a purified organic matrix acidic glycoprotein from mollusk shells behaves in vitro in a similar way.MATERIALS AND METHODS Materials. Untreated polystyrene films (100-,um thick) were obtained from Sterilin (Teddington, Middlesex, England). Poly(aspartic acid) (n = 52, average Mr 6000) was from Bioyeda (Rehovot, Israel) and poly(acrylic acid) (n = 70, average Mr 5000) was from Aldrich. Poly(glutamic acid) (n = 140, average Mr 20,000) was synthesized as described (11). Spectrapor dialysis membranes with Mr cut-off 6000-8000 were from Spectrum Medical Industries (Los Angeles). Falcon cell culture dishes (3 cm in diameter) were obtained from Becton Dickinson. The water used throughout was double-distilled.Polystyrene Films. Polystyrene film...

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Cell adhesion and spreading were studied on sulphonated polystyrene dishes in serum-free saline (Mn, Na, Cl, buffer) i.e., without an intervening protein layer. Spreading as a function of surface charge density, SCD, peaked around 2-10 negative charges per square nanometer, corresponding to a monomolecular layer of sulphonate ions. At optimal SCD, macrophages, BHK-C13 and whole mouse embryo secondary cells all showed considerable spreading, even in monovalent saline-more so than on a conventional tissue-culture surface. But outside this narrow range of SCD, or on protein-coated surfaces, the divalent cation was indispensable. The biphasic effect of sulphonation on cell adhesion is consistent with the theory that a substratum need not be biochemically specific, provided it is physiochemically polar, rigid and dense. According to this theory, polystyrene of sub-optimal SCD would not be sufficiently polar, while supra-optimal sulphonation would produce a hydrogel surface, lacking in local rigidity and density, due to osmotic swelling. The principle of polymer exclusion, by a surface hydrogel layer, is also consistent with observations on the inhibitory effects of adsorbed proteins-viz., albumin, collagen, serum and cellular exudate, respectively-contrasted with the ready attachment of cells to a bare, optimally charged substratum, in this minimal in vitro system.

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