Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite

Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated mechanisms. They have superior mechanical properties due to their complex architecture. Control over biomineral properties can be accomplished by regulation of particle size, shape, crystal orientation, and polymorphic structure. In many organisms, biogenic minerals are assembled using a transient amorphous mineral phase. Here we report that organic constituents of bones and teeth, namely type I collagen and dentin matrix protein 1 (DMP1), are effective crystal modulators. They control nucleation of calcium phosphate polymorphs and the assembly of hierarchically ordered crystalline composite material. Both full-length recombinant DMP1 and post-translationally modified native DMP1 were able to nucleate hydroxyapatite (HAP) in the presence of type I collagen. However, the N-terminal domain of DMP1 (amino acid residues 1-334) inhibited HAP formation and stabilized the amorphous phase that was formed. During the nucleation and growth process, the initially formed metastable amorphous calcium phosphate phase transformed into thermodynamically stable crystalline hydroxyapatite in a precisely controlled manner. The organic matrix-mediated controlled transformation of amorphous calcium phosphate into crystalline HAP was confirmed by x-ray diffraction, selected area electron diffraction pattern, Raman spectroscopy, and elemental analysis. The mechanical properties of the protein-mediated HAP crystals were also determined as they reflect the material structure. Such understanding of biomolecule controls on biomineralization promises new insights into the controlled synthesis of crystalline structures.     

Inhibition of hydroxyapatite crystal growth by bone proteoglycans and proteoglycan components

The small leucine-rich proteoglycans (SLRPs) interact with hydroxyapatite (HAP) and have been demonstrated to be important modulators of mineralisation. In the present study we have examined the effect of bone SLRPs, purified bone glycosaminoglycan (GAG) chains and core proteins as well as commercial chondroitin 4-sulphate, chondroitin 6-sulphate and desulphated chondroitin on HAP crystal growth. Seeded HAP growth experiments revealed that addition of bone GAG chains resulted in almost complete inhibition of crystal growth (93%), with addition of core proteins and intact PGs resulting in 55 and 37% inhibition, respectively. In contrast, commercial chondroitin 4-sulphate was significantly less inhibitory compared with the bone SLRPs and components, yielding only a 6% reduction in HAP-induced crystal growth at the same concentration. Significantly, chondroitin 6-sulphate was found to be noninhibitory, whilst desulphated chondroitin was inhibitory to seeded HAP growth. The data indicate that direct adsorption of SLRPs to growth sites and their ability to bind calcium are significant determinants in the inhibitory process. In addition, PG/GAG chemistry and the conformation of the macromolecules in solution have also been shown to be important. This work provides new information regarding the role of bone SLRPs and their components in the regulation of the mineralisation process.     

Protein nucleation and crystallization by homologous protein thin film template

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.     

Adsorption of chitosan on PET films monitored by quartz crystal microbalance

The adsorption behavior of chitosan on poly(ethylene terephthalate) (PET) model film surface was studied using the quartz crystal microbalance (QCM) technique. QCM with a dissipation unit (QCM-D) represents a very sensitive technique for adsorption studies at the solid/liquid interface in situ, with capability of detecting a submonolayer of adsorbate on the quartz crystal surface. Chitosan as well as PET were chosen for this study due to their promising biocompatible properties and numerous possibilities to be used in biomedical applications. As a first step, PET foils were activated by alkaline hydrolysis in order to increase their hydrophilicity. Model thin films were prepared from PET foils by the spin coating technique. The chemical composition of the obtained model PET films was analyzed using X-ray photoelectron spectroscopy (XPS) and their morphology was characterized by atomic force microscopy (AFM). Furthermore, the adsorption behavior of chitosan on these activated PET films and the influence of adsorption parameters (pH, ionic strength and chitosan solution concentration) were investigated in detail. Additionally, the surface chemistry and morphology of the PET films and the chitosan coated PET films were analyzed with XPS and AFM.     

The effects of serum and human albumin on calcium hydroxyapatite crystal growth.

The effects of potential serum inhibitors upon the growth of calcium hydroxyapatite (HAP) crystals were studied in vivo using a pH-stat system. Whole serum caused a marked decrease in crystal growth in a dose-dependent manner. At a protein concentration of 13 micrograms/ml, whole serum reduced the initial rate of crystal growth from 84 mumol of KOH/h to 48 mumol of KOH/h. Serum components were separated by ultrafiltration (10,000 Da cut-off). The high-molecular-mass fraction containing serum proteins gave an initial rate of crystal growth of 48 mumol of KOH/h compared with 64 mumol of KOH/h given by the low-molecular-mass components. Thus, two-thirds of the inhibitory activity was associated with proteins and other serum macromolecules, whilst the remainder of the activity was associated with the low-molecular-mass components. Albumin-depleted serum showed an initial rate of crystal growth of 59 mumol of KOH/h, whilst albumin purified by affinity chromatography gave an initial rate of crystal growth of 56 mumol of KOH/h at the same protein concentration. Albumin, therefore, not only accounts for half of the protein concentration in serum, but also contributes half of the inhibitory activity of the high-molecular-mass fraction. Heat denaturation of albumin dramatically enhanced the inhibition of HAP seeded growth with the initial rate of crystal growth falling to 27 mumol of KOH/h after treatment compared with 62 mumol of KOH/h before denaturation. Isoelectric focusing indicated that the tertiary and secondary structure, and hence the distribution of surface charge of albumin, are altered by heat denaturation. Gels showed a mixture of species with isoelectric points ranging from 6.0 to 5.0 compared with the native protein value of 4.7. These data suggest that adsorption of serum proteins to the growing HAP crystals is one mechanism of growth inhibition. It is also clear that the most abundant serum protein, albumin, is an important mediator of this process.     

Repair of impurity-poisoned protein crystal surfaces

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.     

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions

Efficient determination of three-dimensional protein structures is critical for unraveling structure-function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (approximately 100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.     

Organization of extracellularly mineralized tissues: a comparative study of biological crystal growth

Biological mineralization processes are extremely diverse and, to date, it is an act of faith rather than an established principle that organisms utilize common mechanisms for forming crystals. A systematic analysis of the structural organization, as far as possible at the molecular level, of five different extracellularly mineralized tissues is presented to demonstrate that at least these mineralization processes are all part of the same continuum. The degrees of control exercised over crystal nucleation and crystal growth modulation are the basic variables. The five tissues, extracellularly mineralizing algae, radial and granular foraminifera, mammalian bone, mammalian enamel, and mollusk shell nacre, probably span the entire spectrum. Their crystal shapes, sizes, and the relations between the mineral phase and the organic phase, are primarily used to assess probable degrees of control exercised over crystal nucleation and modulation. Three different types of nucleation processes can be recognized: nonspecific, stereochemical, and epitaxial. Modulation of crystal growth after nucleation is either absent, achieved by adsorption of macromolecules onto specific crystal faces, or occurs by the prepositioning of matrix surfaces which interrupt crystal growth. The tissues in which active control is exercised over crystal growth all contain similar types of acidic matrix macromolecules. Significantly, the framework matrix macromolecules are all quite different and hence probably perform some tissue-specific functions. The study shows that there is a common basis for understanding these mineralization processes which is reflected in the nature of the protein-crystal interactions which occur in each tissue.     

Nucleation and growth of apatite by a self-assembled polycrystalline bioceramic

The formation aspects of a polycrystalline self-assembled bioceramic leading to the nucleation of hard-tissue mineral from a supersaturated solution are discussed. Scanning electron imaging and surface-sensitive interrogations of the nucleated mineral indicated the presence of an intermediate amorphous layer encompassing a rather crystalline phase that formed on niobium oxide (Nb(2)O(5)) microstructures. The crystalline phase was identified from Raman spectroscopy as hydroxyapatite (HAP), while the phosphorous-rich amorphous layer is suggested to have the chemical form CaO-P(2)O(5). In addition, the mechanism favoring HAP nucleation is discussed in terms of the (0 0 2) and (0 0 1) diffraction planes of HAP and Nb(2)O(5), respectively. The small mismatch along several lattice dimensions strongly suggests epitaxy as a dominant mode in the heterogeneous nucleation of HAP. Furthermore, the effectiveness of this mode was shown to critically depend on the self-organization of the Nb(2)O(5) microstructures. Because nucleation does not appear to depend solely on the integrity of Nb(2)O(5) crystals, the self-organization of Nb(2)O(5) crystals also contributes significantly to HAP nucleation. Based on our results, we propose the organized arrangement of bioceramic crystals as a new mode for the bioinspiration of hydroxyapatite and other hard-tissue mineral.     

Studies of crystal growth mechanisms of proteins by electron microscopy

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.     

Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli

With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by aminotransferase (encoded by aspC). On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid as substrate. To overproduce aspartase in Escherichia coli, the aspA gene was cloned and overexpressed 180 times over the wild-type level. The use of AspA-overproducing E. coli strain for L-Asp production exhibited an 83% conversion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp. Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by using fumaric acid and PPA as substrates. As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively. In sharp contrast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp. This result illustrates a potential and attractive process to yield both L-Asp and L-Phe by coupling AspA and AspC. A further study on the repeated use of the recombinant strain immobilized with calcium alginate showed that after eight batch runs L-Asp conversion maintained roughly constant (around 75%), whereas L-Phe conversion dropped to 65% from 81%. This result indicates the stability of AspA being superior to AspC.     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

Adhesion and growth of HUVEC endothelial cells on films of enzymatically functionalized chitosan with phenolic compounds

Human Umbilical Vein Endothelial Cell (HUVEC) growth on chitosan films and its enzymatically functionalized derivatives films with ferulic acid (FA) and ethyl ferulate (EF) was assessed by evaluating cell adhesion, morphology and cell viability. The results indicated that chitosan derivative films improved protein adsorption properties compared to chitosan films. The HUVEC cell morphology showed well attachment and spread phenotype on chitosan derivative films compared to those growing on chitosan films which did not spread and remained round. Evaluation of cell viability revealed improvement of cell adhesion on chitosan derivative films compared to chitosan film depending on the quantity of oxidized phenols grafted on chitosan. In addition, FA-/EF-chitosan films allowed almost similar cell adhesion. Furthermore, cell adhesion was increased with the film thickness. These results suggested that the oxidized phenols grafting on chitosan is a promising process to enhance cell adhesion, growth and creating useful functional biomaterials.     

The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13C-labeled side chain of Ala46 is 8.0 A from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.     

Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase

Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.     

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.     

Eggshell matrix protein mimics: designer peptides to induce the nucleation of calcite crystal aggregates in solution

Ansocalcin is a novel goose eggshell matrix protein with 132 amino acid residues, which induces the formation of polycrystalline calcite aggregates in in vitro crystallization experiments. The central region of ansocalcin is characterized by the presence of multiplets of charged amino acids. To investigate the specific role of charged amino acid multiplets in the crystal nucleation, three short peptides REWD-16, REWDP-17 (containing charged doublets), and RADA-16 (alternating charged residues) were synthesized and characterized. The aggregation of these peptides in solution was investigated using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering experiments. The peptides REWD-16 and REWDP-17 induced the polycrystalline calcite crystal aggregates, whereas RADA-16 did not induce significant changes in calcite crystal morphology or aggregate formation in in vitro crystallization experiments. The lattice and morphology of the calcite crystals were characterized using X-ray diffraction and scanning electron microscope. The results discussed in this paper reveal the importance of multiplets of charged amino acid residues toward the nucleation of polycrystalline calcite crystal aggregates in solution.     

Calcium carbonate precipitation and crystal morphology induced by microbial carbonic anhydrase and other biological factors

The effect of microbial carbonic anhydrase (CA) on the calcium carbonate (CaCO₃) precipitation was systematically investigated comparing to other biological factors (bovine CA, bovine serum albumin, carboxymethyl chitosan and glutamic acid). The results showed that the precipitation rate of Ca²⁺ in the presence of either microbial CA or bovine CA was faster than that in the presence of 1% bovine serum albumin, 1% carboxymethyl chitosan or 1% glutamic acid, respectively. In addition, XRD analysis indicated that the dominant CaCO₃ crystal phase was calcite. The CaCO₃ crystal morphologies mainly showed cubic and polyhedral shapes induced by microbial CA, and became multiformity induced by other factors from FE-SEM analysis. These results suggested a novel approach for biomimetic synthesis of CaCO₃ materials by microbial CA.     

Probabilistic approach to lysozyme crystal nucleation kinetics

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.     

The contribution of acidulant to the antibacterial activity of acid soluble α- and β-chitosan solutions and their films

This study evaluated individual contributions of dissolving acids (acetic acid, lactic acid, and hydrochloric acid) or acid solubilized chitosan to the antibacterial activity against Listeria innocua and Escherichia coli as solutions and dried films. Solutions containing chitosan showed significantly (P?<?0.05) different inhibitory activity (measured as percentage of inhibition (PI), in percent) against L. innocua and E. coli, compared to equivalent acid solutions. This increase was calculated as additional inhibition (AI, in percent), which could be as high as 65 % in solutions containing 300–320 kDa chitosan depending on the acid type, bacterial species, and the chitosan form (α or β). Solutions containing 4–5 kDa chitosan had lower AI and showed much greater variability among the different chitosan forms, acid types, and bacterial species. Higher molecular weight (Mw) chitosan also showed significantly higher levels of adsorption to bacterial cells than that of lower Mw samples, suggesting that the observed increase in inhibition was the result of surface phenomena. The contribution of acids to the antibacterial activity of chitosan films was assessed by comparing non-rinsed and rinsed films (rinsed in the appropriate broth to remove residual acids and active fragments formed on the dried film). Rinsing β-chitosan films has reduced PI by as much as 28 % compared with non-rinsed films, indicating that part of the antibacterial activity of chitosan films is due to the presence of soluble acid compounds and/or other active fragments. Overall, both acidulant and chitosan were found to contribute to the antibacterial activity of acid solubilized α- and β-chitosan, with the exact antibacterial activity of chitosan varying based on the solution and film properties, suggesting a complex interaction.