Inhibitor Of Hydroxyapatite Crystal Growth Research Articles

Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration

Biomineralization is regulated by inorganic pyrophosphate (PPi), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PPi, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PPi) resulted in increased cementum formation, suggesting PPi metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout (Ank KO), Enpp1 mutant (Enpp1asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 (Ank KO: 8-fold, 3-fold; Enpp1asj/asj: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 (Dmp1/DMP1), osteopontin (Spp1/OPN), and bone sialoprotein (Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PPi modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

Mechanism of inhibition of calcium oxalate crystal growth by an osteopontin phosphopeptide

Osteopontin (OPN) inhibits the nucleation and/or growth of several biominerals, including hydroxyapatite (HA) and calcium oxalate monohydrate (COM), and is thought to function in the prevention of soft-tissue calcification. In previous studies, pOPAR, a peptide corresponding to amino acids 65–80 of rat bone OPN (pSHDHMDDDDDDDDDGD), was shown to be a potent inhibitor of HA crystal growth. We now show that formation of COM in the presence of this peptide results in plate-shaped crystals with rounded ends and scalloped {100} faces. Measurement of crystal dimensions revealed that the pOPAR inhibits growth of COM faces in the order {100} > {121} > {010}. Crystal growth inhibitors are believed to act by adsorbing to growth steps, sites at which lattice-ion addition is energetically favoured. To test this hypothesis, we performed molecular dynamics (MD) simulations of pOPAR adsorption to {121} steps on a {100} face and {121} steps on an {010} face. In the former case, the peptide adsorbs to the {100} (terrace) plane in preference to the {121} (riser) plane; in the latter, the peptide adsorbs to the {121} (riser) plane in preference to the {010} (terrace) plane. These studies represent the first use of MD to study the interaction between an inhibitor and crystal steps. Contrary to the prevailing belief that crystal growth inhibitors adsorb to both lattice planes of a step, we show that pOPAR interacts preferentially to either the terrace or the riser, depending on which is more cationic.

Role for alkaline phosphatase as an inducer of vascular calcification in renal failure?

Vascular calcification is associated with increased cardiovascular morbidity and mortality. A number of calcification inhibitors have been defined recently, including inorganic pyrophosphate (PP(i)), an important physicochemical inhibitor of hydroxyapatite crystal growth. Increased hydrolysis of PP(i) by tissue-nonspecific alkaline phosphatase (TNAP) may occur in renal failure and act to enhance mineralization of vessels.

Effects of pyrophosphate on desmal and endochondral mineralization and TNAP activity in organoid culture

During endochondral and desmal osteogenesis, mineralization of bone and cartilage matrix requires an appropriate solubility product of calcium and phosphate, collagen as a nucleator and deactivation of inhibitors, in order to prevent heterotopic calcification. In the 1960s, Fleisch and coworkers detected pyrophosphate (PPi) as an inhibitor of hydroxyapatite crystal growth, which should be removed by cleavage to tissue non-specific alkaline phosphatase (TNAP) activity. This theory had been established by basic experiments performed with collagen gels and demineralized matrices. In order to investigate the effect of PPi on matrix mineralization in bone and cartilage, calcium content and TNAP activity were measured in organoid cultures of mouse calvarial osteoblasts and limb bud cartilage after treatment with PPi and/or levamisole. In organoid cultures, bone and cartilage develop in a clear histotypical manner. PPi did not induce mineralization. Beta-glycerophosphate (beta-GP) and inorganic phosphate (Pi) induced mineralization which could be significantly reduced by PPi. Levamisole, an inhibitor of TNAP, also reduced mineralization; the combination with PPi was additive. TNAP activity was increased after treatment with PPi and levamisole in both osteoblast and cartilage cultures. Mineralization induced by beta-GP and Pi decreased TNAP activity in the osteoblast but not in cartilage organoid culture. In this culture system, PPi reduced mineralization as predicted by Fleisch's theory. Indications of cleavage of PPi were indirectly found because inhibition of hydrolysis of PPi by levamisole further reduced mineralization, probably due to the higher amounts of PPi available for binding to hydroxyapatite.

Chondrogenesis Mediated by PP i Depletion Promotes Spontaneous Aortic Calcification in NPP1−/− Mice

We recently linked human arterial media calcification of infancy to heritable PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) deficiency. NPP1 hydrolyzes ATP to generate PP(i), a physicochemical inhibitor of hydroxyapatite crystal growth. But pathologic calcification in NPP1 deficiency states is tissue-restricted and in perispinal ligaments is endochondral differentiation-mediated rather than simply a dystrophic process. Because ectopic chondro-osseous differentiation promotes artery calcification in atherosclerosis and other disorders, we tested the hypothesis that NPP1 and PP(i) deficiencies regulate cell phenotype plasticity to promote artery calcification. Using cultured multipotential NPP1-/- mouse bone marrow stromal cells, we demonstrated spontaneous chondrogenesis inhibitable by treatment with exogenous PP(i). We also demonstrated cartilage-specific gene expression, upregulated alkaline phosphatase, decreased expression of the physiological calcification inhibitor osteopontin, and increased calcification in NPP1-/- aortic smooth muscle cells (SMCs). Similar changes were demonstrated in aortic SMCs from ank/ank mice, which are extracellular PP(i)-depleted because of defective ANK transmembrane PP(i) transport activity. Moreover, NPP1-/- and ank/ank mice demonstrated aortic media calcification by von Kossa staining, and intra-aortic cartilage-specific collagen gene expression was demonstrated in situ in NPP1-/- mice. NPP1 and PP(i) deficiencies modulate phenotype plasticity in artery SMCs and chondrogenesis in mesenchymal precursors, thereby stimulating artery calcification by modulating cell differentiation.

Hydroxyapatite binding domains in salivary proteins.

One of the functions of proteins in human saliva is to maintain a balance between calcium phosphate remineralization and demineralization. Saliva is normally supersaturated with respect to calcium phosphate phases such as hydroxyapatite (HAP) and octacalcium phosphate (OCP), but it can also be undersaturated, locally, with respect to these phases (Gron, 1973). The proteins believed to be the strongest precipitation inhibitors are statherin and a group of acidic proline-rich phosphoproteins (PRPs), all of which adsorb strongly to HAP (Moreno et al., 1978, 1979). These molecules contain phosphoserine residues and have a significant number of negatively charged residues localized in their N-terminal regions, whereas the C-termini contain mainly uncharged residues (Schlesinger and Hay, 1977; Wong et al., 1979). The charged domains of both statherin and the PRPs have been shown to have high affinities for HAP surfaces and to be efficient inhibitors of HAP crystal growth in supersaturated solution (Schlesinger et al., 1987; Moreno et al., 1981). However, several other proteins containing charged domains may be moderate or weaker contributors to precipitation inhibition. HAP adsorption and crystallization modification in the presence of a protein depends on the manner in which it adsorbs at the surface. In this study, these data are compared for some salivary proteins.

Properties of phosphorylated 32 kd nonamelogenin proteins isolated from porcine secretory enamel

Enamel proteins were isolated from specific locations of permanent porcine incisors at various developmental stages, namely, the outer (young) and inner (old) secretory, and maturing (chalk-like in appearance) enamel. The selective adsorption of these matrix proteins onto hydroxyapatite (HA) crystals was investigated in the presence of dissociative agents. The results showed that the proteins with the highest adsorption affinity were present at the highest concentration in the vicinity of the ameloblasts, i.e., in the outer enamel layer; a substantial reduction of these proteins was observed in the older (inner) secretory enamel and in the tissue in the maturing stage. An interesting finding was that a group of proteins having molecular masses of 32 kd present only in the inner secretory enamel, adsorbed strongly onto the HA crystals and were potent inhibitors of HA crystal growth. This 32 kd group contains phosphorylated glycoproteins; they are rich in Pro, Glu, Gly, and Asp and the N-terminal sequence was LXQVPGRIPPGYGRPPTP-, having no resemblance to the reported sequences of amelogenins. It was also found that the 32 kd moieties remained only as trace constituents in the maturing enamel, suggesting that most of them were removed as soluble constituents in the tissue fluid or further degraded by enzymatic activity during the late secretory stage. The results obtained support the view that amelogenetic mineralization is regulated by the presence of various organic matter and, importantly, that their efficacy as inhibitors of mineralization may be modulated through their degradation.

The Influence of Salivary Proteins on the Growth, Aggregation and Surface Properties of Hydroxyapatite Particles.

AbstractThe adsorption and crystal growth effects of salivary cystatin SA-II and non-glycosylated amylase on hydroxyapatite have been compared to the effects of the salivary cystatins SA-I and SA-III. Amylase was the least active HAP crystal growth inhibitor and adsorbed weakly to HAP. Although the three cystatins were active inhibitors of hydroxyapatite crystal growth in supersaturated solution, their affinities showed marked differences.

The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion.

The neutral histidine-rich polypeptide (HRP) from human parotid secretion was isolated by ion-exchange and gel-filtration chromatography. The complete amino acid sequence determined by automated Edman degradation of the protein, tryptic and Staphylococcus aureus V8 protease peptides, and digestion with carboxypeptidase A is: (Formula: see text) where Pse represents phosphoserine. The polypeptide contains 38 residues and has Mr 4929. The charged amino acids predominate with 7 histidine, 4 arginine, 3 lysine, 3 aspartic acid, 3 glutamic acid residues, and 1 phosphoserine. Assuming minimal charge contributions from histidine and one negative charge from phosphoserine at pH 7, the net charge of HRP is balanced by an equal contribution of basic and acidic residues. Furthermore, the distribution of hydrophilic and hydrophobic residues along the polypeptide chain indicates that there is no structural polarity. The polypeptide lacks threonine, alanine, valine, cysteine, methionine, and isoleucine. HRP did not display sequence similarity with any protein sequence in the National Biomedical Research Foundation Data Bank. HRP is an active inhibitor of hydroxyapatite crystal growth from solutions supersaturated with respect to calcium phosphate salts and therefore must play a role in the stabilization of mineral-solute interactions in oral fluid. In addition, HRP is a potent inhibitor of Candida albicans germination and therefore may be a significant component of the antimicrobial host defense system in the oral cavity.

Inhibitors of Crystal Growth of Hydroxyapatite: A Constant Composition Approach

Pyrophosphate, citrate and magnesium, inhibitors of hydroxyapatite crystal growth, were studied using a seeded crystal growth system of constant composition at pH 5.80, 6.60 and 7.40. With this technique, crystal growth was studied at constant supersaturation at different pH values without the induction of other calcium phosphate phases. One inhibitor unit (that concentration of material that results in a reduction of 50 per cent in the growth rate from control) was calculated using the Langmuir adsorption isotherm. Pyrophosphate and citrate increased inhibitor activity with decreasing pH, whereas magnesium increased inhibitor activity with increasing pH. These data suggest that, at the urinary concentrations of these inhibitors, pyrophosphate is the most potent inhibitor, citrate less, and magnesium least. Pooled urine collections were studied using the same system and were found to have decreased inhibitor activity as pH decreased. This suggests that other modulators of hydroxyapatite, either promoters or inhibitors, are active in this system at the pH values studied.

Influence of phosphocitrate, a potent inhibitor of hydroxyapatite crystal growth, on mineralization of cartilage and bone

Summary An in vivo bone induction system was used to study the effect of phosphocitrate, a potent inhibitor of calcium phosphate crystallization, on the initial stages of mineralization. Subcutaneous transplantation of coarse powders of demineralized rat diaphyseal bone matrix into allogenic recipients results in a well defined sequence of mineralization steps involving cartilage formation, calcification and bone formation. Phosphocitrate when administered locally or systemically had no effect on the mineralization of either cartilage or bone. Dose levels were chosen to be consistent with that of a previous study which demonstrated that ethane-1-hydroxy-1, 1-diphosphonate (EHDP), a similarly effective in vitro inhibitor of hydroxyapatite crystallization, has a strong inhibitory effect on the initial mineralization steps. Since EHDP is not metabolized in vivo , the lack of effect of phosphocitrate in this study is suggestive of possible enzymatic hydrolysis at the calcification site which may regulate the level of phosphocitrate.

Factors affecting the rate of post-eruptive maturation of dental enamel.

The factors affecting the rate of post-eruptive maturation of dental enamel in Sprague-Dawley rats have been studied, using silver nitrate stain to delineate the hypmineralized areas in molars. Hypomineralization, as measured by the stain, can arise through demineralization, lack of mineralization, or both. Maturation is the net result of the total accumulation of calcium and phosphate in the erupted dental enamel. It was found that the inhibitor of hydroxyapatite crystal growth, disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDPTM)1, applied topically to the newly-erupted molars of rats, decreased the rate of post-eruptive maturation of molars. On the other hand, fluoride (a known stimulator of hydroxyapatite crystal growth and inhibitor of demineralization) applied simultaneously with EHDP restored the rate of post-eruptive maturation of the molars to that of a normal control rat. A dentifrice containing 3% EHDP and 0.1% fluoride significantly reduced the progress of caries in rate. Fluoride treatment alone increased the degree of post-eruptive maturation. Also, in a separate experiment, as the mole ration of EHDP to fluoride was decreased from 16 to 0.5 at constant EHDP level, there was a significant increase in the degree of post-eruptive maturation of dental enamel. It is concluded from the results of this study that although EHDP may inhibit the maturation of rat enamel, fluoride prevents this effect when applied simultaneously. The data support the hypothesis that post-eruptive maturation of dental enamel is governed by the sum of two distinct and opposing processes, mineralization and demineralization. The processes can be inhibited singly or together.