Hydroxyapatite Crystal Growth Research Articles

Histochemical Detection of Zinc in Mineralizing Rat Bone: Autometallographic Tracing of Zinc Ions in the Mineralization Front, Osteocytes, and Osteoblasts

Zinc (Zn) in biological tissue is found in 2 pools. One pool is firmly bound to proteins; ie, Zn is involved in maintaining the 3-dimensional form of molecules or is part of the active site of enzymes. The other pool comprises a pool of free or loosely-bound Zn ions, often stored in vesicles, where they have been found to make peptides and proteins clump.It has been known for some time that bone contains high levels of Zn, whereas, to our knowledge, Zn ions have not previously been histochemically recognized in bone. The aim of the study was, therefore, to detect and localize Zn ions in bone cells and mineralizing bone tissues by the autometallographic (AMG) sulfide silver method.Rats were transcardially perfused with a sodium sulfide solution, whereby nanometer sized Zn sulfide crystals were created at sites containing free or loosely-bound Zn ions. The femora and tibiae were removed and cut on a diamond saw. The 150-μm sections, which included undecalcified cortical bone, were subjected to AMG development, whereby Zn sulfide crystallites were silver-amplified.We found AMG silver grains localized intracellularly in osteoblasts and osteocytes and extracellularly in mineralizing osteoid. Ultrastructurally, the intracellular Zn ions were located in secretory vesicles, and, the extracellular Zn ions were found in matrix vesicles.It has been hypothesized that Zn ions act as an important regulator of calcium influx in matrix vesicles, and their detection inside these vesicles is therefore interesting. During mineralization, Zn ions may participate in the nuclear ization and growth of hydroxyapatite crystals. (The J Histotechnol 22:85, 1999)

Histochemical Detection of Zinc in Mineralizing Rat Bone: Autometallographic Tracing of Zinc Ions in the Mineralization Front, Osteocytes, and Osteoblasts

Zinc (Zn) in biological tissue is found in 2 pools. One pool is firmly bound to proteins; ie, Zn is involved in maintaining the 3-dimensional form of molecules or is part of the active site of enzymes. The other pool comprises a pool of free or loosely-bound Zn ions, often stored in vesicles, where they have been found to make peptides and proteins clump.It has been known for some time that bone contains high levels of Zn, whereas, to our knowledge, Zn ions have not previously been histochemically recognized in bone. The aim of the study was, therefore, to detect and localize Zn ions in bone cells and mineralizing bone tissues by the autometallographic (AMG) sulfide silver method.Rats were transcardially perfused with a sodium sulfide solution, whereby nanometer sized Zn sulfide crystals were created at sites containing free or loosely-bound Zn ions. The femora and tibiae were removed and cut on a diamond saw. The 150-μm sections, which included undecalcified cortical bone, were subjected to AMG development, whereby Zn sulfide crystallites were silver-amplified.We found AMG silver grains localized intracellularly in osteoblasts and osteocytes and extracellularly in mineralizing osteoid. Ultrastructurally, the intracellular Zn ions were located in secretory vesicles, and, the extracellular Zn ions were found in matrix vesicles.It has been hypothesized that Zn ions act as an important regulator of calcium influx in matrix vesicles, and their detection inside these vesicles is therefore interesting. During mineralization, Zn ions may participate in the nuclear ization and growth of hydroxyapatite crystals. (The J Histotechnol 22:85, 1999)

Influence of magnesium substitution on a collagen-apatite biomaterial on the production of a calcifying matrix by human osteoblasts.

The induction of a calcifying matrix is of great interest in the restoration of bone defects. In a previous in vitro study we demonstrated that a collagen sponge constituted of type I collagen fibrils, chondroitin sulfates, and hydroxyapatite crystals induces an earlier and a more abundant synthesis of a new extracellular calcifying matrix than do other biomaterials such as collagen or hydroxyapatite alone. Bone mineral contains various amounts of magnesium ions, either adsorbed at the surface of apatite crystals or incorporated inside the crystal structure. Magnesium is known to reduce the degradation rate of tricalcium phosphate ceramics and to influence the crystallization of mineral substance. Thus we evaluated two sponges modified with different substituted apatites. The substituted low magnesium-containing apatite sample decreased the osteoinductive properties of the sponge whereas the substituted high magnesium-containing apatite sample had a toxic effect on bone cells and prevented the formation of any extracellular matrix. Such a toxic effect can be explained by the presence of large numbers of magnesium ions released into the culture medium even though at physiological level magnesium is able to promote bone mineralization and to control the growth of hydroxyapatite crystals. Thus collagen sponges containing hydroxyapatite remain one of the most appropriately evaluated biomaterials used for the restoration of periodontal pockets and bone defects.

The importance of formation of hydroxyl ions by dissociation of trapped water molecules for growth of calcium hydroxyapatite crystals

The polynuclear mechanism of crystal growth is adapted to describe the growth of calcium hydroxyapatite, HAP. As in the model previously used for growth of fluorapatite, FAP, and dissolution of HAP and FAP, the rate of nucleation is assumed to be a function of mean ion activity, and the lateral growth rate of nuclei is assumed to be controlled by the integration of calcium ions, accompanied by phosphate ions. For growth of HAP, allowance is made for the relatively slow formation of hydroxyl ions from water molecules trapped under the crystal surface. Taking this into account, the frequency for calcium ions to make a diffusion jump into a kink and, simultaneously, to partly dehydrate, calculated from experimental results for growth of HAP, is, within a factor of about 2, in agreement with the theoretical value, 1.6×10 5/s, as found for growth of FAP and dissolution of HAP and FAP. Some misconceptions concerning the interpretation of specific surface areas of crystals are discussed.

Effect of vanadocene dichlorides on the crystal growth of hydroxyapatite

The effect of vanadocenes, used as drugs for various therapeutic applications, on the crystal growth of hydroxyapatite was investigated at sustained supersaturation using the constant composition technique. All vanadocene dichlorides were found to inhibit crystal growth of hydroxyapatite possibly through adsorption onto the active growth sites for crystal growth. The apparent order for the crystallization was n = 2 thus, suggesting a surface-diffusion-controlled mechanism. The kinetic results favoured a Langmuir-type isotherm, suggesting a high affinity of vanadocene dichloride for hydroxyapatite.

Histologic and mechanical evaluation for bone bonding of polymer surfaces grafted with a phosphate-containing polymer

A phosphate-containing polymer was covalently immobilized onto a polyethylene (PE) rod, a poly(ethylene terephthalate) (PET) thread, and a PET film by a surface graft polymerization technique, followed by immersion in calcium phosphate solution to deposit a thin hydroxyapatite (HA) layer on the modified polymer surfaces. The PE rod had a tapered shape, while the PET thread was fixed with clips after implantation, both to minimize the micromovement which may occur in bone. The PE rod was implanted through press-fitting in the femur of rat. Significant enhancement was observed for direct contact of the implant surface with a newly formed bone for both the grafted only and the further HA-deposited PE rods in comparison with the untreated PE at 4, 5, and 6 weeks after implantation. The rats implanted with the modified PET thread in the femur were sacrificed 3 and 6 weeks after implantation. Statistically significant differences were observed for the untreated versus the grafted plus HA-deposited rods at 6 weeks after implantation. To study the resorption of the deposited HA on the methacryloyloxyethylene phosphate-grafted surface, HA-deposited PET films were subcutaneously implanted in the back of rats. The deposited HA was rapidly resorbed within 3 weeks of implantation. These results suggest that the phosphate polymer chains grafted on the PE and PET surfaces effectively induced nucleation and growth of HA crystals. It seems likely that the thin HA layer additionally deposited in vitro onto the grafted PE and PET surfaces was resorbed rapidly and then promoted the growth of HA crystals in vivo.

Histologic and mechanical evaluation for bone bonding of polymer surfaces grafted with a phosphate‐containing polymer

A phosphate-containing polymer was covalently immobilized onto a polyethylene (PE) rod, a poly(ethylene terephthalate) (PET) thread, and a PET film by a surface graft polymerization technique, followed by immersion in calcium phosphate solution to deposit a thin hydroxyapatite (HA) layer on the modified polymer surfaces. The PE rod had a tapered shape, while the PET thread was fixed with clips after implantation, both to minimize the micromovement which may occur in bone. The PE rod was implanted through press-fitting in the femur of rat. Significant enhancement was observed for direct contact of the implant surface with a newly formed bone for both the grafted only and the further HA-deposited PE rods in comparison with the untreated PE at 4, 5, and 6 weeks after implantation. The rats implanted with the modified PET thread in the femur were sacrificed 3 and 6 weeks after implantation. Statistically significant differences were observed for the untreated versus the grafted plus HA-deposited rods at 6 weeks after implantation. To study the resorption of the deposited HA on the methacryloyloxyethylene phosphate-grafted surface, HA-deposited PET films were subcutaneously implanted in the back of rats. The deposited HA was rapidly resorbed within 3 weeks of implantation. These results suggest that the phosphate polymer chains grafted on the PE and PET surfaces effectively induced nucleation and growth of HA crystals. It seems likely that the thin HA layer additionally deposited in vitro onto the grafted PE and PET surfaces was resorbed rapidly and then promoted the growth of HA crystals in vivo. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 384–393, 1997.

Kinetics of nucleation and crystal growth of hydroxyapatite and fluorapatite on titanium oxide surfaces

Although titanium has long been used as an implant material, the mechanisms of its subsequent biomineralization are not yet understood. The nucleation and growth of hydroxyapatite (Ca 5(PO 4) 3OH, HAP) and fluorapatite (Ca 5(PO 4) 3F, FAP) on titanium oxide (TiO 2) surfaces have been investigated using constant composition (CC) methods in supersatured solutions at 37°C and ionic strength 0.15 mol l −1 with relative supersaturation, 13.4–15.0 and 13.5–16.5 for HAP and FAP, respectively. The solid phases during the reaction were examined by X-ray diffraction, scanning electron microscopy (SEM) and diffuse reflectance Fourier transform infrared spectroscopy (FTIR). Both crystal phases nucleated at the TiO 2 particle surfaces with supersaturation-dependent induction periods, τ in, preceding the initial crystal growth, varying from 8 to 17 h for HAP and from 7 to 15 h for FAP. The interfacial tensions, γ, estimated both from initial growth rate data following nucleation and from the induction times were about 32 and 39 mJ m −2 for FAP and HAP, respectively. These values were of the same order of magnitude as those determined by a contact angle method involving thin layer techniques. The results show that TiO 2 will nucleate both HAP and FAP, but only after relatively long induction periods. It is also interesting to note that the X-ray diffraction pattern of HAP on TiO 2 surfaces resembles that of bone and root dentin mineral in the region 2θ from 30 to 35°.

Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins

Phosphoproteins in human saliva include proline-rich proteins, statherins, histatin I and cystatin SA-III. The presence of phosphate in these proteins is necessary for various functions in the mouth including calcium binding, inhibition of precipitation of calcium phosphate, inhibition of growth of hydroxyapatite crystals and adherence to hydroxyapatite. To elucidate the process of phosphorylation of these proteins, the phosphorylation of a peptide (APRP8) with an amino acid sequence identical to one of the phosphorylated sites in acidic proline-rich proteins by a kinase from the human sublingual gland was investigated. The kinase, which was highly labile, was purified 58-fold by fractionation of sublingual gland homogenate and gel filtration, but the enzyme was inactivated when further purification by chromatographic techniques commonly used for protein kinases was attempted. To compare the enzyme with other kinases, and to obtain information that could be used in its further purification, a characterization was undertaken. The enzyme required 10 MM Mg 2+ for optimum activity, it had a K M of 0.09 mM for ATP and the KM for the peptide substrate APRP8 was 0.42 mM. It was not activated by cAMP or calmodulin, characteristics that are shared with casein kinases and mammary gland kinase. The sublingual kinase as well as casein kinase 2 were inhibited by heparin, but in other respects the two kinases had different properties. While casein kinase 2 is activated by polylysine and has optimal activity in 150 mM KCI, sublingual kinase was inhibited by polylysine and the addition of KCI. Moreover, casein kinase 2 can utilize both ATP and GTP as phosphoryl donors, but GTP was not a substrate for sublingual kinase. The sublingual kinase shared a substrate recognition sequence with mammary gland kinase, but, unlike that kinase, it could not utilize Cat` instead of Mgt 2+. While the sublingual kinase thus shared some properties with both casein kinase 2 and mammary gland kinase, distinct differences were also seen and the relationship to these enzymes remains to be determined. The characterization of the sublingual kinase will be useful in its further purification.

Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain.

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.

Influence of polymers for use in saliva substitutes on de- and remineralization of enamel in vitro.

A number of polymers which have previously been tested for their applicability as thickening agents in saliva substitutes were studied in vitro for their caries-protective properties. These were: polyacrylic acid, carboxymethylcellulose, xanthan gum, guar gum, hydroxyethylcellulose and porcine gastric mucin. The polymers were tested for their effects on: (1) growth of hydroxyapatite crystals in a supersaturated calcium phosphate solution, (2) dissolution of hydroxyapatite crystals in 50 mM acetic acid, pH 5.2 and (3) demineralization and remineralization of bovine enamel in a pH-cycling model. Growth of hydroxyapatite crystals was strongly inhibited by polyacrylic acid and carboxymethylcellulose at very low concentrations (0.005% w/v). Other polymers displayed lower inhibition of hydroxyapatite crystal growth. Hydroxyapatite dissolution was inhibited by all polymers except by hydroxymethylcellulose and xanthan gum. This occurred both in the presence of the polymers as well as after a 30-min preincubation. In the pH-cycling experiment, bovine enamel specimens with preformed lesions were alternately exposed to a demineralization buffer and a remineralization buffer containing the polymers hydroxyethylcellulose, carboxymethylcellulose, xanthan gum, polyacrylic acid, or porcine gastric mucin. A remineralization buffer containing 1 ppm NaF was used as a positive control. Under the experimental conditions, the control experiment without additives resulted in a net mineral loss (30.6 mumol Ca/cm2 after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 mumol/cm2). All polymers largely inhibited further demineralization (1.2-12.3 mumol/cm2) except polyacrylic acid which, inhibited of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 mumol/cm2). In conclusion, polymers tested in this study, except the polyacrylic acid, reduced the demineralization of enamel in vitro. The precise mechanism of the protective effect is not clear but it is speculated that formation of an absorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.

Measurement of hydroxyapatite crystal growth rate by phase shift interferometry

Measurement of hydroxyapatite crystal growth rate by phase shift interferometry

P59. Modulation of hydroxyapatite crystal growth: the role of albumin and endogenous matrix in enamel development

P59. Modulation of hydroxyapatite crystal growth: the role of albumin and endogenous matrix in enamel development

Influence of phospholipid on bile salt binding to calcium hydroxyapatite and on the poisoning of nascent hydroxyapatite crystals.

Glycine-conjugated, dihydroxy bile salts inhibit calcium hydroxyapatite (HAP) formation by binding to and poisoning nascent crystal embryos. Their taurine-conjugated counterparts bind less well to hydroxyapatite and do not inhibit its formation; but more hydrophobic, synthetic analogs of the taurine conjugated bile salts are inhibitors of hydroxyapatite formation. Because hydrophobicity is an important determinant of the ability of bile salts to inhibit hydroxyapatite crystal growth, experiments were performed to study the effect of the physiologically important mixed micelles of bile salt and phospholipid. Taurodeoxycholate/phosphatidylcholine (10:1) mixed micelles bound to HAP at lower total lipid concentrations than did pure taurodeoxycholate. At low total lipid concentrations, phosphatidylcholine (PC) binding appeared to predominate, suggesting that PC had a higher affinity than did taurodeoxycholate (TDC) for the HAP surface. Although glycodeoxycholate (3 mM) significantly (> 95%) inhibited hydroxyapatite precipitation, higher concentrations of taurodeoxycholate, either alone or mixed with phosphatidylcholine, did not affect hydroxyapatite formation. These results suggest that biliary phospholipids do not modulate the ability of bile salts to inhibit hydroxyapatite crystal growth.

Precipitation of Hydroxyapatite under Stearic Acid Monolayers

The nucleation and growth of hydroxyapatite (HAp) crystals from solutions with and without Langmuir monolayers of stearic acid (SA) were investigated. The monolayers apparently enhanced the nucleation of HAp. Monolithic, filmlike polycrystalline HAp with preferred orientation of (00l) planes parallel to the monolayer interface was precipitated, while colloidal and powdery precipitation in the bulk solution was predominant in the absence of the monolayer. Geometrical similarity was found between the hexagonal packing conformation of the SA headgroups in the monolayers and the alignment of calcium ions in the (001) plane of HAp. It was suggested that HAp crystals nucleated and grew epitaxially on the monolayer interface.

Influence of synthetic salivary cystatin SN segments on hydroxyapatite mineralization

Three segments of cystatin SN (CsnSN) have been synthesized which contain possible hydroxyapatite (HAP) binding sites. The influence of the fragments on HAP crystal growth has been studied using the constant composition (CC) method in solutions with a relative supersaturation with respect to HAP ( σ HAP) of 3.6, pH 7.4, and 0.15 mol l −1 ionic strength. The additives studied were CsnSN fragments containing residues 1–20, 21–40, and 82–101. At molar surface concentrations of (2–3) × 10 −7 mol m −2, the two N-terminal CsnSN fragments inhibited HAP growth to a similar extent while the C-terminal segment of comparable size was less effective as a crystallization inhibitor. This indicates a more effective HAP surface coverage by CsnSN(1–20) and CsnSN(21–40) and that their charged residues are located in more favourable positions than CsnSN(82–101) for binding to the HAP surfaces. Also, relative inhibitory effects of salivary proteins and their fragments suggest that the degree to which an inhibitor changes the growth rates is also influenced by the nature of the rate-controlling crystal growth mechanism.

Effect of Two New Polysaccharides on Growth, Agglomeration and Zeta Potential of Calcium Phosphate Crystals

Purpose To study the effect of semisynthetic sulphated polysaccharides in the different calcium phosphate crystallization processes in vitro. Materials and Methods Crystallization of hydroxyapatite (HAP) and brushite (DCPD) in the presence and absence of 2 new semisynthetic sulphated polysaccharides (G871, G872) were defined by a constant composition technique, particle size analysis and zeta potential measurement. Results These polysaccharides demonstrated strong inhibitory effect on HAP and DCPD crystal growth and agglomeration. The increase of negative zeta potential values after addition of polysaccharides suggests the binding of these polysaccharides to HAP and DCPD crystals. Conclusion We conclude that both G871 and G872 could be of potential use for calcium phosphate urolithiasis prevention in addition to their use for calcium oxalate.

The Dual Role of Fibrinogen as Inhibitor and Nucleator of Calcium Phosphate Phases: The Importance of Structure

Constant composition and free drift methods have been used to investigate the abilities of human serum albumin (HSA) and fibrinogen to influence calcium phosphate precipitation. Both molecules inhibit hydroxyapatite (HAP) crystal growth when present in the solution phase. Fibrinogen, when immobilized at hydrophobicized germanium or silica surfaces, is able to nucleate calcium phosphate phases; at clean germanium or silica surfaces, it appears to be inactive. The apparent configuration of fibrinogen molecules at germanium or silica surfaces on which octadecyltrichlorosilane (OTS) was deposited probably exposes more negative sites for participation in nucleation.

Cluster model for hydroxyapatite crystal growth

Cluster model for hydroxyapatite crystal growth

Fluoride Modulates the Inhibition of in Vitro Hydroxyapatite Crystal Growth by Small Dentin Proteoglycan: Relevance to Dental Calculus

Glycosaminoglycan constituents of the periodontium have been detected in supragingival and subgingival dental calculus. They are polyanionic heteropolysaccharides containing -COOH and SO3H residues. The non-sulfated hyaluronan is present in supragingival calculus, whereas chondroitin sulfate, dermatan sulfate, and heparan sulfate are detected in subgingival calculus. They are implicated in both ectopic and endogenous mineralization, and have been used in the present study to investigate seeded hydroxyapatite crystal growth, and the influence of fluoride on the process. All glycosaminoglycans examined inhibited crystal growth, with chondroitin-4-sulfate and dermatan sulfate being more effective than the hyaluronan. Fluoride alone enhanced growth and reduced the inhibitory influence of the glycosaminoglycans and the parent proteoglycan, which is a potent inhibitor. The results yield important information on the role of fluoride and proteoglycan in ectopic mineralization.