Biomimetic calcium phosphate mineralization with multifunctional elastin-like recombinamers

Biomimetic hybrid materials based on a polymeric and an inorganic component such as calcium phosphate are potentially useful for bone repair. The current study reports on a new approach toward biomimetic hybrid materials using a set of recombinamers (recombinant protein materials obtained from a synthetic gene) as crystallization additive for calcium phosphate. The recombinamers contain elements from elastin, an elastic structural protein, and statherin, a salivary protein. Via genetic engineering, the basic elastin sequence was modified with the SN(A)15 domain of statherin, whose interaction with calcium phosphate is well-established. These new materials retain the biocompatibility, "smart" nature, and desired mechanical behavior of the elastin-like recombinamer (ELR) family. Mineralization in simulated body fluid (SBF) in the presence of these recombinamers reveals surprising differences. Two of the polymers inhibit calcium phosphate deposition (although they contain the statherin segment). In contrast, the third polymer, which has a triblock structure, efficiently controls the calcium phosphate formation, yielding spherical hydroxyapatite (HAP) nanoparticles with diameters from 1 to 3 nm after 1 week in SBF at 37 °C. However, at lower temperatures, no precipitation is observed with any of the polymers. The data thus suggest that the molecular design of ELRs containing statherin segments and the selection of an appropriate polymer structure are key parameters to obtain functional materials for the development of intelligent systems for hard tissue engineering and subsequent in vivo applications.     

Synthesis of soluble phosphate polymers by RAFT and their in vitro mineralization

Soluble linear (non-cross-linked) poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were successfully synthesized through reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization and by keeping the molecular weight below 20 K. Above this molecular weight, insoluble (cross-linked) polymers were observed, postulated to be due to residual diene (cross-linkable) monomers formed during purification of the monomers, MOEP and MAEP. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy)ethyl methacrylate) (PAAEMA) were successfully prepared and were immobilized on aminated slides. Simulated body fluid studies revealed that calcium phosphate (CaP) minerals formed on both the soluble polymers and the cross-linked gels were very similar. Both the PMAEP polymers and the PMOEP gel showed a CaP layer most probably brushite or monetite based on the Ca/P ratios. A secondary CaP mineral growth with a typical hydroxyapatite (HAP) globular morphology was found on the PMOEP gel. The soluble PMOEP film formed carbonated HAP according to Fourier transform infrared (FTIR) spectroscopy. Block copolymers attached to aminated slides showed only patchy mineralization, possibly due to the ionic interaction of negatively charged phosphate groups and protonated amines.     

Fabrication of gelatin/calcium phosphate composite nanofibrous membranes by biomimetic mineralization

Based on the principles of biomimetic mineralization, biocomposite nanofibrous membranes were fabricated by the growth of CaP crystals on electrospun gelatin nanofibers to mimic both the physical architecture and chemical composition of natural bone ECM. Plenty more CaP crystals formed on the nanofibrous membrane containing Ca(2+) ion precursors, in which these crystals were also observed on the inner side of membrane. The release rate of Ca(2+) ion precursors from the nanofibrous membrane was slower than that of PO(4)(3-) ion precursors, suggesting the existence of more strong intermolecular interaction between gelatin and Ca(2+) ions. ATR-FTIR and XRD results clearly revealed the formation of CaP crystals mixed with apatite and CaCO(3), or apatite and TCP on the membranes. The Ca/P molar ratio of crystals obtained from the XPS data was 2.03 and 1.60, which depended on the mineralization conditions. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane, meaning to the improved in vivo bone bioactivity.     

Polymer brush controlled bioinspired calcium phosphate mineralization and bone cell growth

Polymer brushes on thiol-modified gold surfaces were synthesized by using terminal thiol groups for the surface-initiated free radical polymerization of methacrylic acid and dimethylaminoethyl methacrylate, respectively. Atomic force microscopy shows that the resulting poly(methacrylic acid) (PMAA) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes are homogeneous. Contact angle measurements show that the brushes are pH-responsive and can reversibly be protonated and deprotonated. Mineralization of the brushes with calcium phosphate at different pH yields homogeneously mineralized surfaces, and preosteoblastic cells proliferate on both the nonmineralized and mineralized surfaces. The number of living cells on the mineralized hybrid surfaces is ca. 3 times (PDMAEMA) and 10 times (PMAA) higher than on the corresponding nonmineralized brushes.     

Protein cocrystallization molecules originating from in vitro selected macrocyclic peptides

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Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.     

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGNE 6 transfection technology. The nocovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a mximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G₂/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.     

Regulation of vitamin D metabolism by calcium and phosphate ions in isolated renal tubules.

The acute and long-term effects of Ca2+ and Pi on vitamin D metabolism were studied in vitro with isolated renal tubules from vitamin D-deficient and vitamin D-supplemented chicks. Ca2+ depletion, achieved by isolating renal tubules in Ca2+-free buffers, led to suppression of 1 alpha-hydroxylase activity. Re-introduction of Ca2+ during incubation caused an acute stimulation of this enzyme. This stimulatory effect of Ca2+ was prevented by prior treatment of Ca2+-depleted renal tubules for 6 h with 1,25-dihydroxycholecalciferol. Ca2+ and Pi produced marked acute affects on 1 alpha-hydroxylase activity, which persisted for the whole 8 h experimental period, in Ca2+-depleted renal tubules from vitamin D-deficient chicks. The effects of either ion were influenced by the concentration of the other. However, the effects of these ions could not be reproduced in either Ca2+-depleted renal tubules from vitamin D-supplemented chicks or in renal tubules from vitamin D-deficient chicks, isolated in Ca2+-containing buffers. Isolation of renal tubules from vitamin D-supplemented chicks in Ca2+-containing buffers and subsequent incubation for 8 h in the presence of increased [Ca2+] led to a modest but statistically significant suppression of 1 alpha-hydroxylase and stimulation of 24-hydroxylase activity. It is concluded that the acute effects of Ca2+ and Pi on 1 alpha-hydroxylase activity of Ca2+-depleted renal tubules from vitamin D-deficient chicks are not regulatory but the results of the experimental conditions. In contrast the long-term effects of Ca2+ on both hydroxylases of renal tubules from vitamin D-supplemented chicks may be of physiological significance.     

Precipitation of nucleotides by calcium phosphate

Earlier it has been shown that nucleic acids of high molecular weight can be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5′-monophosphate were coprecipitated to 29–87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2′p5′A2′p5′A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.     

Regulation of apoptosis by vasoactive peptides

Although originally discovered because of their ability to affect hemodynamics, vasoactive peptides have been found to function in a variety of capacities including neurotransmission, endocrine functions, and the regulation of cell proliferation. A growing body of evidence describes the ability of vasoactive peptides to regulate cell death by apoptosis in either a positive or negative fashion depending on the peptide and the type of target cell. The available evidence to date is strongest for the peptides endothelin, angiotensin II, vasoactive intestinal peptide, atrial natriuretic peptide, and adrenomedullin. Each of these peptides is discussed, with specific regard to apoptosis, in terms of regulatory activity, target cell specificity, and potential role in pulmonary physiology.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues

$\text{In vitro}$ effects of toxaphene on Ca²⁺-ATPase activity and ⁴⁵Ca²⁺-uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca²⁺-ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca²⁺-ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca²⁺-ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial ⁴⁵Ca²⁺-uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca²⁺-ATPase and calcium transport in heart, kidney and liver tissues of rat.     

Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes

Mg²⁺ and Zn²⁺ are present in the mineral of matrix vesicles (MVs) and biological apatites, and are known to influence the onset and progression of mineral formation by amorphous calcium phosphate (ACP) and hydroxyapatite (HAP). However, neither has been studied systematically for its effect on mineral formation by phosphatidylserine-Ca²⁺-Pi complexes (PS-CPLX), an important constituent of the MV nucleation core. Presented here are studies on the effects of increasing levels of Mg²⁺ and Zn²⁺ on the process of mineral formation, either when present in synthetic cartilage lymph (SCL), or when incorporated during the formation of PS-CPLX. Pure HAP and PS-CPLX proved to be powerful nucleators, but ACP took much longer to induce mineral formation. In SCL, Mg²⁺ and Zn²⁺ had significantly different inhibitory effects on the onset and amount of mineral formation; HAP and PS-CPLX were less affected than ACP. Mg²⁺ and Zn²⁺ caused similar reductions in the rate and length of rapid mineral formation, but Zn²⁺ was a more potent inhibitor on a molar basis. When incorporated into PS-CPLX, Mg²⁺ and Zn²⁺ caused significantly different effects than when present in SCL. Even low, subphysiological levels of Mg²⁺ altered the inherent structure of PS-CPLX and markedly reduced its ability to induce and propagate mineral formation. Incorporated Zn²⁺ caused significantly less effect, low (<20 μM) levels causing almost no inhibition. Levels of Zn²⁺ present in MVs do not appear to inhibit their nucleational activity.     

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca^2 +) and inorganic phosphate (P_i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca^2 + and P_i transport are limited. The aim of the present study was to characterize the intestinal Ca^2 + and P_i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca^2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca^2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca^2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D_9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca^2 + transport. Regarding the P_i transport, the results indicate significant active P_i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na⁺/K⁺-ATPase on the serosal-to-mucosal P_i transport suggests a pivotal role of Na⁺ in jejunal P_i transport in the horse.     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.     

Short-time pre-washing of brushite-forming calcium phosphate cement improves its in vitro cytocompatibility

A pre-washing protocol was developed for resorbable, brushite-forming calcium phosphate cements (CPCs) to avoid harmful in vitro effects on cells. CPC discs (JectOS+, Kasios; self-developed CPC) were pre-washed with repeated changes of phosphate-buffered saline (PBS; 24 h total). Unwashed or PBS-pre-washed discs were incubated in culture medium (5% fetal calf serum; up to 10 days) and then tested for their influence on pH/calcium/phosphate levels in H₂O extracts. Effects on pH/calcium/phosphate levels in culture supernatants, and morphology, adherence, number, and viability of ATDC5 cells and adipose-tissue derived stem cells were analyzed in co-culture. Pre-washing did not alter CPC surface morphology or Ca/P ratio (scanning electron microscopy; energy-dispersive X-ray spectroscopy). However, acidic pH of unwashed JectOS+ and self-developed CPC (5.82; 5.11), and high concentrations of Ca (2.17; 2.40 mM) and PO₄ (38.15; 49.28 mM) in H₂O extracts were significantly counteracted by PBS-pre-washing (pH: 7.92; 7.92; Ca: 0.64; 1.11 mM; PO₄: 5.39–5.97 mM). Also, PBS-pre-washing led to physiological pH (approx. 7.5) and PO₄ levels (max. 5 mM), and sub-medium Ca levels (0.5–1 mM) in supernatants and normalized cell morphology, adherence, number, and viability. This CPC pre-washing protocol improves in vitro co-culture conditions without influencing its structure or chemical composition.     

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.     

Regulation of nebramycin biosynthesis by inorganic phosphate

The effect of inorganic phosphate on the biosynthesis of nebramycin factors2, 4 and5′ was studied inStreptomyces tenebrarius strain A (forming2, 4 and5′ in natural ratios) and its mutants B (forming predominantly2), C (forming2 as the only major product) and D (forming predominantly5′). In phosphate-supplemented complex media, the production of2 in A, B and C was reduced by 20–70%, while the yields of5′ remained unchanged in A and decreased by 30–60% in B. The production of4 increased by 50–90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.     

Ultrastructural evidence in vitro of osteoclast-induced degradation of calcium phosphate ceramic by simultaneous resorption and phagocytosis mechanisms

Osteoclasts are physiological polykaryons specialized in the resorption of calcified tissue. In the context of the clinical use of calcium-phosphate (CaP) ceramics as bone substitutes, this study used transmission electron microscopy to investigate the in vitro mechanisms of CaP ceramic degradation by osteoclastic cell types. Osteoclasts cultured on CaP ceramic developed typical ultrastructural features of bone osteoclasts, such as a polarized dome shape, a clear zone and a ruffled border. Modification of the shape and density of CaP crystals under the ruffled border indicated an acidic microenvironment. Moreover, osteoclasts were able to degrade ceramic by simultaneous resorption and phagocytosis mechanisms. Phagocytosis did not alter the ability of osteoclasts to resorb CaP ceramic. The phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. The common mechanism of phagocytosis described here is similar to that observed with the monocyte/macrophage lineage, confirming that osteoclasts are part of the mononuclear phagocyte system. Osteoclasts are thus clearly involved in CaP degradation by means of resorption and phagocytosis.