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High pressure effects on the colloidal calcium phosphate and the structural integrity of micellar casein milk. Part 1. High pressure dissolution of colloidal calcium phosphate in heated milk systems.

Results of this study confirm that high temperature (118 degrees C/15 min) and high pressure (400 MPa/5 min) processing of skim milk, skim milk ultrafiltration and ultracentrifugation fractions, and model milk salt solutions cause dramatic shifts in their colloidal and soluble Ca phosphate equilibrium that affect their pH, dissolved Ca content, turbidity, and casein micelle microstructure. The relations between high temperature and high pressure processing-induced changes in the colloidal and soluble Ca phosphate equilibrium were evaluated in raw, pasteurized, and high temperature treated skim milk, ultrafiltration retentate and permeate of pasteurized skim milk, clear ultracentrifugation infranatant of pasteurized skim milk, and synthetic milk ultrafiltrates with and without lactose or Ca. The magnitude of the pH and dissolved Ca shifts caused by high temperature and high pressure processing was a function of casein micelle concentration. Ultrafiltration permeate exhibited the most drastic shits in pH and dissolved Ca contents due to high temperature and high pressure processing. Although high temperature processing reduced the pH of ultrafiltration permeate from 6.59 to 6.03 and the dissolved Ca from 100% to 58%, high pressure processing reversed both of these changes. These changes in high temperature and high pressure processed milk, milk fractions, and model milk salt solutions were related to microstructural changes in the casein micelles as revealed by electron microscopy.

Animals↗

[Research development and prospect of calcium phosphate biomaterials with intrinsic osteoinductivity].

Calcium phosphates ceramics are frequently used as bone substitute materials in clinics because they are similar to the bone in mineral phase, they are of no antigenicity, and they have good biocompatibility as well as excellent osteoconductivity. The question as to whether calcium phosphate biomaterials possess osteoinductivity has been debated for a long time. Up to now, extensive fundamental studies and clinical application have demonstrated that calcium phosphate ceramics with special structure may induce bone formation in soft tissue. In this paper are reviewed the discovery and confirmation of calcium phosphates ceramics with intrinsic osteoinductivity, the process and mechanism of osteoinduction, and the relationship between osteoinductivity of calcium phosphate materials and animal species, the mesenchymal stem cells related to osteoinduction, the application of osteoinductive biomaterials. The researches directed toward to the osteoinduction of calcium phosphate are prospected.

Biocompatible Materials↗

Fiber reinforced calcium phosphate cement.

The term calcium phosphate cement was introduced by Gruninger et al. (1). This type of cement can be prepared by reacting a calcium phosphate salt with an aqueous solution, which causes it to set by the crossing of the precipitated crystals. These cements offer a series of advantages that allow their use as grafts and substitutes of damaged parts of the bone system. However, these cements have low mechanical strength compared to human bones. This work studied the influence of the use of polyamide fibers in the mechanical properties of a calcium phosphate cement based on alpha-tricalcium phosphate as well as the mechanisms involved in the increase of mechanical strength. The results demonstrate the feasibility of the use of polymeric fibers to increase mechanical strength and the need for coupling agents for the effective performance of the fibers as reinforcement in these materials.

Adsorption↗

Preparation and characterization of an electrodeposited calcium phosphate coating associated with a calcium alginate matrix.

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.

Alginates↗

Reactions involving carbamyl phosphate in the presence of precipitated calcium phosphate with formation of pyrophosphate: a model for primitive energy-conservation pathways.

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.

Adenosine Monophosphate↗

Induction of early growth response gene Egr2 by basic calcium phosphate crystals through a calcium-dependent protein kinase C-independent p44/42 mitogen-activated protein kinase pathway.

Using the reverse transcriptase-polymerase chain reaction we examined the effect of basic calcium phosphate (BCP) crystals on the induction of the early growth response gene Egr2 transcription and the signal transduction pathway involved. The results showed that BCP crystals induced Egr2 transcription up to 8-fold, peaking at 24 h after treatment. The induction of Egr2 was confirmed by transient transfection assays using an Egr2 promoter/luciferase reporter construct and could be inhibited by the p44/42 mitogen-activated protein kinase (MAPK)-specific inhibitor U0126, or by calcium chelator TMB-8, but not by the SAPK2/p38 MAPK inhibitor SB202190 or by the protein kinase C inhibitor bisindolylmaleimide I (Bis-I). Using the Mercury Pathway Profiling System (Clontech, Palo Alto, Calif., USA) we further showed that induced Egr2 could stimulate the activities of several transcription factors that are associated with cell proliferation, such as c-fos, SRF and c-myc.

Animals↗

Molecular mechanism of the induction of metalloproteinases 1 and 3 in human fibroblasts by basic calcium phosphate crystals. Role of calcium-dependent protein kinase C alpha.

Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration. These crystals are mitogenic and induce oncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts. To date, BCP crystal-elicited signal transduction pathways have not been completely studied. Because protein kinase C (PKC) is known to play an important role in signal transduction, we investigated the participation of this pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent PKC signal transduction pathway and that the PKC alpha isozyme is specifically involved in the pathway. We have previously shown that BCP crystal induction of MMP-1 and MMP-3 is also dependent on the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We now show that these two pathways operate independently and seem to complement each other. This leads to our hypothesis that the two pathways initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.

Base Sequence↗

[Calcium phosphate ceramics in orthopedic surgery].

Calcium phosphate ceramics might replace bone grafts in orthopaedic surgery. The chemical determination of these materials is now well known, and world-wide animal experiments as well as our own experience have proven that these products are both biocompatible and bioactive. The early chemical and physico-chemical mechanisms of interaction between calcium phosphate ceramics and bone tissue and biological fluids at the site of implantation are numerous (degradation, dissolution, crystal precipitation). These processes precede the cellular mechanisms of osteogenesis which take place during contact with the materials and define osteoconduction. When implanted in a bony environment, the ceramic ensures the formation of a genuine bone which within a few months will acquire all the characteristics of a normal lamellated bone with areas of apposition and resorption during remodeling. However, the mechanical characteristics of calcium phosphate ceramics do not permit an early loading, and they should only be used to fill bone cavities. In vertebral arthrodesis they must be implanted in stress-free areas and combined with strong and stable appliances. To benefit from the special bioactivity of calcium phosphate ceramics, research workers are now turning towards the treatment of metal alloys or composite fibres surfaces as well as of calcium phosphate complexes and extracellular matrix constituents (collagen, proteoglycans, etc.).

Biocompatible Materials↗

Characterization of the adsorption of conjugated and unconjugated bile acids to insoluble, amorphous calcium phosphate.

Recently we showed that supplemental dietary calcium stimulates the intestinal formation of insoluble calcium phosphate and decreases the ratio of dihydroxy to trihydroxy bile acids in human duodenal bile. Because previous in vitro studies indicated that these effects could be due to differential adsorption of bile acids to amorphous calcium phosphate, we characterized the binding of bile acids to calcium phosphate. Freshly formed, amorphous, calcium phosphate bound and thus precipitated glycine-conjugated and unconjugated bile acids, whereas taurine-conjugated bile acids showed little binding. Glycochenodeoxycholic acid hardly adsorbed to other insoluble calcium phosphates, including hydroxyapatite. Adsorption studies using increasing amounts of glycine-conjugated and unconjugated bile acids showed that binding occurred above a bile acid-specific critical minimum concentration, dependent on bile acid hydrophobicity. The simultaneous use of a fluorescent hydrophobic probe indicated that this binding was due to ionic adsorption of monomers of bile acids, followed by their hydrophobic aggregation on the calcium phosphate surface, probably in the form of a bilayer. Finally, using human duodenal bile we found that amorphous calcium phosphate, but not Ca2+, preferentially bound and thus precipitated dihydroxy bile acids. We conclude that freshly formed, amorphous, calcium phosphate is a prerequisite for adsorption of bile acids and that monomers of glycine-conjugated and unconjugated dihydroxy bile acids have a high binding affinity for amorphous calcium phosphate.

Adsorption↗

The fabrication and biochemical evaluation of alumina reinforced calcium phosphate porous implants.

Alumina reinforced calcium phosphate porous implants were manufactured to improve the mechanical strength while maintaining the bioactivity of calcium phosphate ceramics. The alumina porous bodies, which provided the mechanical strength, were fabricated by a polyurethane sponge method and multiple coating techniques resulted in the porous bodies with a 90-75% porosity and a compressive strength of up to approximately 6MPa. The coating of hydroxyapatite (HAp) or tricalcium phosphate (beta-TCP) was performed by dipping the alumina porous bodies into calcium phosphate ceramic slurries and sintering the specimens. The fairly strong bonding between the HAp or TCP coating layer and the alumina substrate was obtained by repeating the coating and sintering processes. The biochemical evaluations of the porous implants were conducted by in vitro and in vivo tests. For in vitro test, the implants were immersed in Ringer's solution and the release of Ca and P ions were detected and compared with those of calcium phosphate powders. For in vivo test, the porous bodies were implanted into mixed breed dogs and bone mineral density measurements and histological studies were conducted. The alumina reinforced HAp porous implants had a higher strength than the HAp porous implants and exhibited a similar bioactivity and osteoconduction property to the HAp porous implants.

Aluminum Oxide↗

Effect of heat treatment on pulsed laser deposited amorphous calcium phosphate coatings.

Amorphous calcium phosphate coatings were produced by pulsed laser deposition from targets of nonstoichiometric hydroxyapatite (Ca/P = 1.70) at a low substrate temperature of 300 degrees C. They were heated in air at different temperatures: 300, 450, 525 and 650 degrees C. Chemical and structural analyses of these coatings were performed using X-ray diffraction (XRD), FTIR, and SEM, XRD analysis of the as-deposited and heated coatings revealed that their crystallinity improved as heat treatment temperature increased. The main phase was apatitic, with some beta-tricalcium phosphate in the coatings heated at 525 and 600 degrees C. In the apatitic phase there was some carbonate substitution for phosphate and hydroxyl ions at 450 degrees C and almost solely for phosphate at 525 and 600 degrees C as identified by FTIR. This was accompanied by a higher hydroxyl content at 525 and 600 degrees C. At 450 degrees C a texture on the coating surface was observable by SEM that was attributable to a calcium hydroxide and calcite formation by XRD. These phases almost disappeared at 600 degrees C, probably due to a transformation into calcium oxide.

Calcium Phosphates↗

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of several human calculi containing calcium phosphate crystals.

Human calcium phosphate calculi: two sialoliths, a urolith, a rhinolith, and a tonsillolith were investigated by scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDX). The sialoliths and urolith had appositional shells with thick cortices, respectively, around several nuclei composed of calcospherulites and a rubber-film fragment. The rhinolith had a thin cortex with appositional laminations around a glomerulus-like mass of calcified cotton-like strings. The tonsillolith had a rough cortex with appositional laminations. Its porous interior was composed of numerous calcified conglomerates with microorganisms and calcified masses with fine appositional laminations around the conglomerates. The major crystals were identified as biological apatites (AP) with a sand-grain rather than a needle-like shape, and plate-shaped octacalcium phosphate (OCP). The AP deposits of the rhinolith probably were associated with magnesium (Mg) phosphates or contained Mg. No OCP was found in the rhinolith. The AP deposits were mainly formed by extracellular calcification. Hexahedral crystals, identified as Mg-containing whitlockite (WH), were precipitated in the internal spaces of the AP and OCP deposits. The rhinolith nucleus consisted of WH crystal deposits only.

Adult↗

Arthropathies associated with basic calcium phosphate crystals.

Basic calcium phosphate (BCP) crystals refer to a family of crystals including partially carbonate substituted hydroxyapatite, octacalcium phosphate, and tricalcium phosphate. These crystals have been found in and around joints and have been associated with several forms of arthritis and periarthritis. Identification of BCP crystals remains problematic because of the lack of a simple, reliable analytic procedure. Methods currently in use include alizarin red S staining, labelled diphosphonate binding, scanning and transmission electron microscopy with energy dispersive X-ray microanalysis, X-ray diffraction, and atomic force microscopy. Periarthropathies associated with BCP crystals include calcific tendinitis and bursitis. Intra-articular BCP crystal deposition is common in osteoarthritis, often found together with calcium pyrophosphate dihydrate crystals. Uncommon conditions in which BCP crystals are found include destructive shoulder arthropathies, acute inflammatory attacks of arthritis, and erosive arthritis. Secondary deposition of BCP crystals has been observed in chronic renal failure, in patients with "collagen vascular" diseases, following neurologic injury and after local corticosteroid injection.

Arthritis↗

Comparison of osteoblast responses to hydroxyapatite and hydroxyapatite/soluble calcium phosphate composites.

Hydroxyapatite/soluble calcium phosphate composites (HAp/SCaP) are novel HAp-based materials with enhanced solubility that have been developed by annealing HAp in a vacuum. This study compared the effects of HAp and HAp/SCaP on osteoblast proliferation, differentiation, and mineralization using an MC3T3-E1 cell culture system. MC3T3-E1 cells were cultured on HAp or HAp/SCaP, and the number of attached cells and their morphology were examined. The influence of the extract from HAp/SCaP on osteoblast differentiation was determined by the measurement of alkaline phosphatase activity and reverse transcriptase-polymerase chain reaction analysis of the expression of osteoblastic markers. In addition, mineralization was evaluated by the staining of calcium deposits with Alizarin red. Attachment of a greater number of cells exhibiting no degeneration in their morphology was observed on HAp/SCaP compared with HAp after incubation for 7 days or more. Culturing cells with the extract from HAp/SCaP resulted in promotion of alkaline phosphatase activity, the expression of type I collagen, and bone-like tissue formation. The results of the present study indicate that HAp/SCaP shows greater ability in osteogenesis than HAp by increasing collagen synthesis and calcification of the extracellular matrix.

Alkaline Phosphatase↗

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings.

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.

Calcium Phosphates↗

In vivo study of a calcium phosphate cement consisting of alpha-tricalcium phosphate/dicalcium phosphate dibasic/tetracalcium phosphate monoxide.

Prehardened calcium phosphate cement consisting of alpha-tricalcium phosphate (alpha-TCP), dicalcium phosphate dibasic (DCPD) and tetracalcium phosphate monoxide (TeCP) was implanted in rabbit mandibles and back muscles, and studied histologically and microradiographically. In the mandibles, new bone formation occurred around the implants and increased in quantity the longer the implant period lasted. Histology, microradiography and scanning electron microscopy (SEM) demonstrated direct contact of bone and cement. Bone response to this cement was essentially the same as to hydroxyapatite (HA) ceramics, known as a biocompatible bone substitute. Material resorption was recognized, which increased with the implant period and was greater in the surface bound by soft tissue than the surface bound by bone tissue. In the back muscles, however, no calcified tissue formation occurred. Resorption proved to be faster than in the case of the mandible implants. It was concluded that the cement, in prehardened form, has good biocompatibility and is a promising material as a bone substitute.

Animals↗

Effects of hydroxypropyl methylcellulose and other gelling agents on the handling properties of calcium phosphate cement.

The calcium phosphate cement (CPC) used in this study was formed by combining equimolar amounts of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA). This powder, when mixed with water, sets to a hard cement in about 30 min. However, the water-based CPC paste is not highly cohesive and is vulnerable to washout until hardening occurs. The objectives of this study were to investigate the effects on handling properties, washout resistance, cement hardening behavior, and mechanical properties of adding several gelling agents to CPC paste. Aqueous solutions that contained a mass fraction of 2-4% of hydroxypropyl methylcellulose (HPMC), carboxyl methylcellulose (CMC), chitosan acetate, and chitosan lactate were used as cement liquids. Hardening time was measured by the Gilmore needle test; resistance to washout was evaluated by the disintegration of the cement specimen in water with agitation; and mechanical strength was evaluated by the measurement of diametral tensile strength and compressive strength. Handling properties were greatly improved by the addition of HPMC, CMC, chitosan acetate, and chitosan lactate. Hardening time was retarded by the additions of HPMC and CMC, and mechanical strength was weakened by the addition of either the chitosan lactate or the chitosan acetate.

Biocompatible Materials↗

Injectable and macroporous calcium phosphate cement scaffold.

Calcium phosphate cement (CPC) can be molded and self-hardens in vivo to form resorbable hydroxyapatite with excellent osteoconductivity. The objective of this study was to develop an injectable, macroporous and strong CPC, and to investigate the effects of porogen and absorbable fibers. Water-soluble mannitol was used as porogen and mixed with CPC at mass fractions from 0% to 50%. CPC with 0-40% mannitol was fully extruded under a syringe force of 10 N. The paste with 50% mannitol required a 100-N force which extruded only 66% of the paste. At fiber volume fraction of 0-5%, the paste was completely extruded. However, at 6% and 7.5% fibers, some fibers were left in the syringe after the paste was extruded. The injectable CPC scaffold had a flexural strength (mean+/-sd; n=5) of (3.2+/-1.0) MPa, which approached the reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, the injectability of a ceramic scaffold, a macroporous CPC, was studies for the first time. Processing parameters were tailored to achieve high injectability, macroporosity, and strength. The injectable and strong CPC scaffold may be useful in surgical sites that are not freely accessible by open surgery or when using minimally invasive techniques.

Biomechanical Phenomena↗