Calcium Phosphate Crystal Growth Research Articles

Enhydra fluctuans Lour. aqueous extract inhibited the growth of calcium phosphate crystals: An in vitro study

Enhydra fluctuans Lour. is consumed by various tribes in their ethnomedicinal practices for the treatment of kidney stones and urinary problems. However, no scientific studies were conducted to evaluate its effect on crystal growth. Hence, the present study proposed to investigate the effect of aqueous extract of whole plant of standardized E. fluctuans (AEEF) on the growth of calcium phosphate (brushite) crystals. Attempts were also made to evaluate the effect of in vitro free radical scavenging and antimicrobial activities of AEEF. In vitro studies indicated that AEEF (50, 100, and 200 µg/mL) exhibited an inhibitory role on brushite crystal growth. The average length of the deposited brushite crystals was decreased by citric acid (1molL−1) and AEEF (200, 100, and 50 µg/mL) till day 8. Results showed that AEEF (200 and 100 µg/mL) and citric acid significantly (P<0.001) decreased the crystal bed as compared to the control on day 8 of the study. AEEF showed an antimicrobial effect against Staphylococcus aureus and exhibited antioxidant activity. In conclusion, the aqueous extract of the whole plant of Enhydra fluctuans was found effective in the inhibition of brushite crystals. Further, in vivo studies along with molecular studies and bioactivity-guided fractionation are required to strengthen its antilithiatic effect along with identifying the bioactive compounds and the mechanism of action involved therein.

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions.

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Biochemical Fulvic Acid Modification for Phosphate Crystal Inhibition in Water and Fertilizer Integration.

Biochemical fulvic acid (BFA), produced by organic wastes composting, is the complex organic matter with various functional groups. A novel modified biochemical fulvic acid (MBFA) which possessed stronger chelating ability had been synthesized by the grafting copolymerization of BFA and acrylic acid (AA). Results showed that MBFA effectively inhibited the crystallization of calcium phosphate and increased the concentration of phosphate in water solution. The optimum reaction conditions optimized by Box–Behnken design and response surface methodology were reaction temperature 69.24 °C, the mass of monomer to fulvic acid ratio 0.713, the initiator dosage 19.78%, and phosphate crystal-inhibition extent was 96.89%. IR spectra demonstrated AA was grafted onto BFA. XRD data and SEM images appeared the formation and growth of calcium phosphate crystals was effectively inhibited by MBFA.

Effect of Convection on Crystal Growth of Calcium Phosphate in a Thermostat under Terrestrial and Space Conditions

Mass transfer in the process of crystal growth of octacalcium phosphate Ca8H2(PO4)6 and hydroxyapatite Ca10(OH)2(PO4)6 during mixing aqueous solutions of CaCl2 and KH2PO4 + K2HPO4 in a buffer solution KCl is simulated mathematically with regard to the stoichiometric relationship. The investigations are carried out for the weightlessness, microgravity, and normal gravity conditions. The effect of gravity (convection) on the nature of reaction component transfer in the process of crystal growth of calcium phosphate under the temperature–controlled conditions is considered. It is shown that the reaction component transfer times are less under convection mixing as compared with the purely diffusive regime, but the rate and mass of formation of calcium phosphate depend not only on the convection time and intensity but also on the convection mixing structure.

The interaction of sodium trimetaphosphate with collagen I induces conformational change and mineralization that prevents collagenase proteolytic attack

ObjectivesThis study evaluated the cell viability and expression of different major genes involved in mineralization in odontoblast-like cells exposed to sodium trimetaphosphate (STMP). It was also investigated the influence of STMP on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization. MethodsImmortalized rat odontoblast MDPC-23 cells were cultured. Cell viability was assessed by trypan blue staining, and the changes in gene expression balance induced by STMP were assessed by quantitative reverse transcription (qRT) PCR assays. Crystalline particle formation was monitored by light-scattering detectors to estimate pH variation and the radial size of the crystalline particles as a function of reaction time (pH 7.4, 25°C) in the presence of STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under atomic force microscopy (AFM) to measure the particle size in the presence of STMP. A three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams after immersion in STMP solution. The binding mechanism of STMP to collagen fibrils and potential stabilization mechanism was assessed with circular dichroism spectrometry (CD). The data were analyzed statistically (α=0.05). ResultsSTMP had no significant influence on the cell viability and gene expression of the MDPC-23 cells. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM-treated crystal size. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen. SignificanceThe increased resistance of collagen against the proteolytic activity of collagenases appears to be related to the conformational change induced by STMP binding in collagen I and the STMP capacity for promoting biomimetic mineralization in type I collagen fibrils.

Particle-Attachment-Mediated and Matrix/Lattice-Guided Enamel Apatite Crystal Growth.

Tooth enamel is a hard yet resilient biomaterial that derives its unique mechanical properties from decussating bundles of apatite crystals. To understand enamel crystal nucleation and growth at a nanoscale level and to minimize preparation artifacts, the developing mouse enamel matrix was imaged in situ using graphene liquid cells and atomic resolution scanning transmission electron and cryo-fracture electron microscopy. We report that 1-2 nm diameter mineral precipitates aggregated to form larger 5 nm particle assemblies within ameloblast secretory vesicles or annular organic matrix subunits. Further evidence for the fusion of 1-2 nm mineral precipitates into 5 nm mineral aggregates via particle attachment was provided by matrix-mediated calcium phosphate crystal growth studies. As a next step, aggregated particles organized into rows of 3-10 subunits and developed lattice suprastructures with 0.34 nm gridline spacings corresponding to the (002) planes of apatite crystals. Mineral lattice suprastructures superseded closely matched organic matrix patterns, suggestive of a combination of organic/inorganic templates guiding apatite crystal growth. Upon assembly of 2-5 nm subunits into crystal ribbons, lattice fringes indicative of the presence of larger ordered crystallites were observed surrounding elongating crystal ribbons, presumably guiding the c-axis growth of composite apatite crystals. Cryo-fracture micrographs revealed reticular networks of an organic matrix on the surface of elongating enamel crystal ribbons, suggesting that protein coats facilitate c-axis apatite crystal growth. Together, these data demonstrate (i) the involvement of particle attachment in enamel crystal nucleation, (ii) a combination of matrix- and lattice-guided crystal growth, and (iii) fusion of individual crystals via a mechanism similar to Ostwald ripening.

Effect of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/gelatin ratios on the characteristics of biomimetic composite nanofibrous scaffolds

Biomimetic composite nanofibrous scaffolds were fabricated via the growth of calcium phosphate (CaP) crystals on electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/gelatin (PHGE) nanofibers with different polymer ratio to mimic the composite nature of bone tissue as well as the nanoscale features of extracellular matrix (ECM). The resulting composite scaffolds exhibited three-dimensionally interconnected microporous structures. The CaP crystals were successfully formed on not only the external surface but also the interior of the scaffolds. The amount of CaP crystals fabricated and the surface roughness of the scaffolds increased with increasing the PHBV content because of the formation of bead-typed CaP aggregates. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane. The results of cytocompatibility tests demonstrated that PHGE41 scaffold, composed of PHBV/gelatin (4:1), promoted more rapid MC3T3-E1 proliferation and differentiation compared with other scaffolds. These results suggest that the PHGE composite scaffolds are ideal biomaterials for bone tissue engineering.

Magnesium Counteracts Vascular Calcification

Over the last decade, an increasing number of studies report a close relationship between serum magnesium concentration and cardiovascular disease risk in the general population. In end-stage renal disease, an association was found between serum magnesium and survival. Hypomagnesemia was identified as a strong predictor for cardiovascular disease in these patients. A substantial body of in vitro and in vivo studies has identified a protective role for magnesium in vascular calcification. However, the precise mechanisms and its contribution to cardiovascular protection remain unclear. There are currently 2 leading hypotheses: first, magnesium may bind phosphate and delay calcium phosphate crystal growth in the circulation, thereby passively interfering with calcium phosphate deposition in the vessel wall. Second, magnesium may regulate vascular smooth muscle cell transdifferentiation toward an osteogenic phenotype by active cellular modulation of factors associated with calcification. Here, the data supporting these major hypotheses are reviewed. The literature supports both a passive inorganic phosphate–buffering role reducing hydroxyapatite formation and an active cell-mediated role, directly targeting vascular smooth muscle transdifferentiation. However, current evidence relies on basic experimental designs that are often insufficient to delineate the underlying mechanisms. The field requires more advanced experimental design, including determination of intracellular magnesium concentrations and the identification of the molecular players that regulate magnesium concentrations in vascular smooth muscle cells.

Correlation of Salivary Statherin and Calcium Levels with Dental Calculus Formation: A Preliminary Study.

Background Salivary constituents have a wide range of functions including oral calcium homeostasis. Salivary proteins such as statherin inhibit crystal growth of calcium phosphate in supersaturated solutions and interact with several oral bacteria to adsorb on hydroxyapatite. Concurrently, saliva, which is supersaturated with respect to calcium phosphates, is the driving force for plaque mineralization and formation of calculus. Thus, the aim of the present study was to estimate and correlate salivary statherin and calcium concentration to the dental calculus formation. Methods A cross-sectional study was conducted to assess the relationship between salivary statherin, calcium, and dental calculus among 70 subjects, aged 20–55 years. Subjects were divided into 3 groups based on the calculus scores as interpreted by Calculus Index which was followed by collection of whole saliva using Super•SAL™. Salivary calcium levels were assessed by calorimetric method using Calcium Assay kit (Cayman Chemical, Michigan, USA) and statherin levels by using ELISA Kit (Cusabio Biotech). Results Statherin levels showed a weak negative correlation with the calcium levels and with calculus formation. The mean salivary statherin and calcium concentration were found to be 0.96 μg/ml and 3.87 mg/ml, respectively. Salivary statherin levels differed significantly among the three groups (p < 0.05). Conclusions Our preliminary data indicates that statherin could possibly play a role in the formation of dental calculus.

Hydrothermal fabrication and characterization of calcium phosphate anhydrous/chitosan composites

Calcium phosphate-based biomaterials are of special interest in bone regeneration due to their biocompatibility and biodegradability; on the other hand, chitosan has novel properties such as antibacterial properties, biocompatibility, and biodegradability. In the present study, a new calcium phosphate (CaP)/chitosan (Chi) nanocomposite biomaterial has been introduced, which was prepared using in situ precipitation by the hydrothermal method. The fabricated nanocomposites were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and electrochemical method. The results showed that crystalline calcium phosphate nanoparticles were mineralized in chitosan and the interaction was formed between the phosphate ion in CaP and positively charged functional groups in chitosan molecular chains. The X-ray diffraction pattern of the synthesized powder showed monetite as the major phase. The effect of chitosan on the crystallization of calcium phosphate particles was studied, and it was confirmed that the polymer has a controlling role over the growth of calcium phosphate crystals and morphologies.

Dimensionally and morphologically controlled growth of calcium phosphate crystals by an organic-free hydrothermal process

Calcium phosphate (CaP) materials are optimal candidate biomaterials for bone tissue engineering because of their bioactivity and biocompatibility. The controlled fabrication of CaP materials draws great interest because the size, shape and composition of CaP crystals play critical roles in biological response. In this study, CaP nanowires and microspheres were synthesised via an organic-free hydrothermal process without the assistance of any surfactant or template. The effect of the reaction condition on CaP crystal growth was investigated. Experimental results showed that the size, shape and composition of the obtained CaP crystals could be manipulated by changing nucleation temperature, reaction temperature, pH value and reaction time. The chemical composition of the obtained crystals had a common change process from dicalcium phosphate dihydrate and dicalcium phosphate anhydrous to hydroxyapatite with the elongation of reaction time. The prenucleation and nucleation temperature before hydrothermal treatment were critical for the growth of CaP crystal. Two typical morphologies of nanowire and microsphere were fabricated under the given reaction condition. The growth mechanisms of the crystals were also discussed in this paper. In vitro results showed that both nanowires and microspheres of CaP exhibited exceptional performance to improve osteoblast proliferation, which endowed them with interesting potential for bone repair. The findings are highly important to develop excellent materials suitable for bone tissue engineering.

A mesoporous biomaterial for biomimetic crystallization in dentinal tubules without impairing the bonding of a self-etch resin to dentin.

CaCO3@mesoporous silica reacted with phosphoric acid (denoted as CCMS-HP) enables the growth of calcium phosphate crystals in dentinal tubules. This study tested whether CCMS-HP could be used to form a biomimetic barrier on the exposed dentin for prevention of dentin sensitivity without impairing the bonding of Single Bond Universal (SBU) self-etch adhesive to the dentin. Twenty-four dentin disks were prepared and divided into three groups: (1) SBU group (n=8), in which SBU self-etch adhesive was bonded to the dentin disk directly; (2) CCMS-HP group (n=8), in which CCMS-HP was applied onto the dentin surface; and (3) CCMS-HP/SBU group (n=8), in which the dentin surface was first treated with CCMS-HP and then boned by SBU. The permeation depth of crystals into the dentinal tubules was examined and measured with a scanning electron microscope. The shear bonding strength of SBU and CCMS-HP/SBU to dentin was also measured. The mean crystal permeation depth was 35.8±6.9μm for the CCMS-HP/SBU group and 33.6±12.2μm for the CCMS-HP group; no significant difference was found between the two groups. Moreover, the mean shear bonding strength was 22.7±6.7MPa for the CCMS-HP/SBU group and 23.3±7.0MPa for the SBU group. There was also no significant difference between the two groups. CCMS-HP can be used to form a biomimetic barrier for prevention of dentin sensitivity because it neither impedes the bonding of SBU to dentin nor impairs the shear bonding strength between the SBU and dentin.

Fractal Measure and Microscopic Modeling of Osseointegration.

In this study, the process of osseointegration on titanium implant surfaces with different physicochemical treatments subjected to a simulated corporal fluid submersion was evaluated using the concept of fractal dimension. It was found that different treatments led to rather different calcium phosphate crystal growth patterns, with fractal dimension ranging from 1.68 to 1.93. The observed crystal patterns may be explained by a general deposition, diffusion, and aggregation growth mechanism, where diffusing particle sticking probability plays a fundamental role.

Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs.

Role of Nanoscale Confinement on Calcium Phosphate Formation at High Supersaturation

An important factor controlling bone mineralization is spatial restriction within collagen fibrils and matrix vesicles. Such nanoscale confinements in addition to chemical control restrict the growth and final size of calcium phosphate crystals (CaP). To study the role of spatial control on CaP mineralization exclusively, we investigated formation of CaPs under high supersaturations (0.84 M Ca2+ and 2.1 M Ca2+) within ordered aqueous domains (8–27 nm) of polymerized liquid crystals (PLCs). The mineralization of CaPs was achieved by a pressurized gas induced pH rise. CaPs mineralized within varying degrees of confinements showed contrasting results. Confinements ≤10 nm mediated the formation of CaPs to phase pure calcium apatite (CaAp), while confinements between 11 and 27 nm resulted in a mixture of phases such as CaAp, amorphous calcium phosphates (ACP), and acidic polymorphs such as brushite and monetite. Results indicated that smaller confinements offer a low particle/solution interface effectively forming small nucleation clusters leading to a metastable ACP phase. We suggest that CaP formation from high supersaturations strongly depends on the degree of confinement around the nucleation site to efficiently control the mineral phase purity. Moreover, a possible insight from this study is that synthetic and biological confinements, in addition to spatial restriction, also rely on chemical control to obtain phase pure CaPs such as bone apatite.

The expanded amelogenin polyproline region preferentially binds to apatite versus carbonate and promotes apatite crystal elongation.

The transition from invertebrate calcium carbonate-based calcite and aragonite exo- and endoskeletons to the calcium phosphate-based vertebrate backbones and jaws composed of microscopic hydroxyapatite crystals is one of the great revolutions in the evolution of terrestrial organisms. To identify potential factors that might have played a role in such a transition, three key domains of the vertebrate tooth enamel protein amelogenin were probed for calcium mineral/protein interactions and their ability to promote calcium phosphate and calcium carbonate crystal growth. Under calcium phosphate crystal growth conditions, only the carboxy-terminus augmented polyproline repeat peptide, but not the N-terminal peptide nor the polyproline repeat peptide alone, promoted the formation of thin and parallel crystallites resembling those of bone and initial enamel. In contrast, under calcium carbonate crystal growth conditions, all three amelogenin-derived polypeptides caused calcium carbonate to form fused crystalline conglomerates. When examined for long-term crystal growth, polyproline repeat peptides of increasing length promoted the growth of shorter calcium carbonate crystals with broader basis, contrary to the positive correlation between polyproline repeat element length and apatite mineralization published earlier. To determine whether the positive correlation between polyproline repeat element length and apatite crystal growth versus the inverse correlation between polyproline repeat length and calcium carbonate crystal growth were related to the binding affinity of the polyproline domain to either apatite or carbonate, a parallel series of calcium carbonate and calcium phosphate/apatite protein binding studies was conducted. These studies demonstrated a remarkable binding affinity between the augmented amelogenin polyproline repeat region and calcium phosphates, and almost no binding to calcium carbonates. In contrast, the amelogenin N-terminus bound to both carbonate and apatite, but preferentially to calcium carbonate. Together, these studies highlight the specific binding affinity of the augmented amelogenin polyproline repeat region to calcium phosphates versus calcium carbonate, and its unique role in the growth of thin apatite crystals as they occur in vertebrate biominerals. Our data suggest that the rise of apatite-based biominerals in vertebrates might have been facilitated by a rapid evolution of specialized polyproline repeat proteins flanked by a charged domain, resulting in apatite crystals with reduced width, increased length, and tailored biomechanical properties.

Rapid Biomimetic Mineralization of Hydroxyapatite-g-PDLLA Hybrid Microspheres

Hydroxyapatite-graft-poly(D,L-lactide) (HA-g-PDLLA) nanoparticles were synthesized here to fabricate hybrid microspheres with diameter in the range of 150-200 μm by emulsion solvent evaporation techniques. The as-obtained microspheres were treated with alkaline solution in order to selectively degrade the PDLLA layer which covered on the surface of hybrid microspheres and instead to generate a dense coating of HA nanoparticles. The hybrid microspheres with enriched HA nanoparticles on the surface were further immersed in simulated body fluid (SBF) solution to evaluate the bone-forming ability of the bioactive hybrid microspheres via the in vitro biomimetic mineralization process. The resultant microspheres were analyzed by using X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA) to understand the nucleation and growth of bioactive calcium phosphate (Ca-P) crystals as a function of surface treatment. Results in this work clearly demonstrated that the existing HA nanoparticles on the surface of hybrid microspheres after alkaline treatment greatly affect the growth of the bone-like Ca-P crystals in SBF solutions. The biomimetic hybrid microspheres were found to be excellent candidates for use as injectable scaffolds for bone tissue engineering.

Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells

Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization.

Laser-Assisted Biomimetic Process for Calcium Phosphate Coating on a Hydroxyapatite Ceramic

The present authors recently developed a new calcium phosphate (CaP) coating technique on an ethylene-vinyl alcohol copolymer substrate utilizing a laser-assisted biomimetic (LAB) process. In the present study, the LAB process was applied to a sintered hydroxyapatite (sHA) substrate for CaP coating. The LAB process was carried out by irradiating the sHA substrate immersed in a supersaturated CaP solution with a low-energy Nd-YAG pulsed laser. Within 30 min of irradiation, contiuous CaP layers with different morphologies were successfully formed on the laser-irradiated sHA surface. A submicron cavernous structure of the CaP layer was developed into a micron flake-like structure as the laser power increased from 1 to 3 W. This result suggests that the secondary nucleation and growth of CaP crystals were accelerated by laser irradiation in a power-dependent manner. Laser absorption by the sHA substrate and the resulting increase in ambient temperature locally near the surface should be responsible for the accelerated CaP nucleation and growth. The present CaP coating technique using the LAB process is simple and quick, hence it would be useful in orthopedic and dental applications as an on-demand surface-functionalization method for biomaterials consisting of sHA.

Mouse bone marrow‐derived mesenchymal stem cell response to nanostructured titanium substrates produced by high‐pressure torsion

Mesenchymal stem cells (MSCs) with the ability to differentiate into various mesoderm‐like cells are known to migrate to various organs to repair injured tissues. They can attach to the implant surface, differentiate into bone‐forming cells, and ultimately osseointegrate with the prosthesis. This study investigates bone marrow‐derived mesenchymal stem cellular response to the grain structure of titanium substrates produced by high‐pressure torsion and annealing processes. Cell attachment, proliferation, viability, and morphology are evaluated on the surface of differently processed nanostructured and coarse‐grained samples. The bacterial adhesion and calcium phosphate crystal formation and growth are also assessed on the surface of the substrates. The nanostructured titanium shows significantly higher cell adhesion, proliferation, spreading, and viability compared with the untreated and coarse‐grained titanium substrates. The adhesion of bacteria is lower and surface bioactivity is higher on the surface of the nanostructured titanium substrate. The results demonstrate the superior MSC compatibility, antibacterial efficacy, and surface bioactivity of the nanostructured titanium substrates, which could lead to early implant fixation and improved osseointegration. Copyright © 2012 John Wiley &amp; Sons, Ltd.