Amorphous Calcium Phosphate Phase Research Articles

A Kinetic Model for Hydroxyapatite Precipitation in Mineralizing Solutions

Numerous studies have shown that there is an amorphous calcium phosphate (ACP) phase preceding the precipitation of crystalline hydroxyapatite (HA) in calcium phosphate solutions. It has also been shown that the addition of magnesium to the solutions has a stabilizing effect by inhibiting the transformation of ACP to HA. The stabilizing effect of Mg2+ is attributed to the stronger bonds between water molecules and the magnesium ions adsorbed on the surface of the ACP particles, making it harder for them to dehydrate. However, the kinetics of the reactions between calcium and phosphate ions to form ACP and then HA crystals, and the effects of varying concentrations of Mg on the kinetics have not been studied theoretically in detail. In this study, we develop and validate a kinetic model for analyzing such reactions. The pertinent rate constants are derived by calibrating the model against temporal changes in Ca2+ concentration reported by others. The predicted onset and growth of HA crystallization for sol...

Synthesis of Magnesium Doped Amorphous Calcium Phosphate as a Bioceramic for Biomedical Application: In Vitro Study

In this study, the effect of addition of magnesium as a stabilizer of amorphous phase on the structure and bioactivity of amorphous calcium phosphate for biomedical applications was investigated. Therefore, amorphous calcium phosphate was synthesized by a precipitation and freeze drying method with different molar ratios of magnesium to calcium (Mg/Ca). Then the samples were incubated in simulated body fluid (SBF) for 3, 7 and 14 days to evaluate their bioactivity and the investigation of their phase conversion. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy were employed to characterize the prepared samples. According to X-ray diffraction patterns, the most stable amorphous phase was obtained at a magnesium to calcium (Mg/Ca) ratio of 0.3 and the magnesium acted as a stabilizer of the amorphous phase. After incubation in SBF solution, the hydroxyapatite phase was detected in XRD patterns of the samples, but with the addition of the magnesium, the crystallinity and formation of the hydroxyapatite phase was decreased. Also the FTIR analysis showed that the intensity of the peaks related to phosphate and hydroxyl groups decreased with the addition of magnesium, which indicated a more stable amorphous calcium phosphate phase. Finally, in-vitro experiments like MTT assay and alkaline phosphatase activity with human osteosarcoma cell lines (G-292) on the prepared samples were evaluated and discussed. The results indicated that amorphous calcium phosphate with a calcium to magnesium ratio of 0.3 revealed optimal cell proliferation and ALP activity. Eventually, this material is non-toxic and compatible to be used as a bone substitute material.

Proton Environments in Biomimetic Calcium Phosphates Formed from Mesoporous Bioactive CaO-SiO2-P2O5 Glasses in Vitro: Insights from Solid-State NMR.

When exposed to body fluids, mesoporous bioactive glasses (MBGs) of the CaO–SiO2–P2O5 system develop a bone-bonding surface layer that initially consists of amorphous calcium phosphate (ACP), which transforms into hydroxy-carbonate apatite (HCA) with a very similar composition as bone/dentin mineral. Information from various 1H-based solid-state nuclear magnetic resonance (NMR) experiments was combined to elucidate the evolution of the proton speciations both at the MBG surface and within each ACP/HCA constituent of the biomimetic phosphate layer formed when each of three MBGs with distinct Ca, Si, and P contents was immersed in a simulated body fluid (SBF) for variable periods between 15 min and 30 days. Directly excited magic-angle-spinning (MAS) 1H NMR spectra mainly reflect the MBG component, whose surface is rich in water and silanol (SiOH) moieties. Double-quantum–single-quantum correlation 1H NMR experimentation at fast MAS revealed their interatomic proximities. The comparatively minor H species of each ACP and HCA component were probed selectively by heteronuclear 1H–31P NMR experimentation. The initially prevailing ACP phase comprises H2O and “nonapatitic” HPO42–/PO43– groups, whereas for prolonged MBG soaking over days, a well-progressed ACP → HCA transformation was evidenced by a dominating O1H resonance from HCA. We show that 1H-detected 1H → 31P cross-polarization NMR is markedly more sensitive than utilizing powder X-ray diffraction or 31P NMR for detecting the onset of HCA formation, notably so for P-bearing (M)BGs. In relation to the long-standing controversy as to whether bone mineral comprises ACP and/or forms via an ACP precursor, we discuss a recently accepted structural core–shell picture of both synthetic and biological HCA, highlighting the close relationship between the disordered surface layer and ACP.

S53P4 Bioactive Glass - an Alternative Treatment of Bone Defects

A challenging problem in orthopedic practice is represented by bone defects may they occur from trauma, malignancy, infection or congenital disease. Bioactive Glasses have a widely recognized ability to foster the growth of bone cells, and to bond strongly with both hard and soft tissues. Upon implantation, Bioactive Glasses undergoes specific reactions, leading to the formation of an amorphous calcium phosphate or crystalline hydroxyapatite phase on the surface of the glass, which is responsible for its strong bonding with the surrounding tissue. This phenomenon sustains a more rapid healing of bone defects and presents great antibacterial properties. In this paper we report on a clinical study that uses S53P4 Bioactive Glass to successfully treat bone defects and testify of the good compatibility of this material with human tissues.

Cracking caused by cutting of plasma-sprayed hydroxyapatite coatings and its relation to the structural features of coatings deposited at different initial substrate temperatures

The present study estimated the cracking phenomenon in as-plasma-sprayed hydroxylapatite coatings (HACs) after they were being subjected to the severe cutting conditions in the direction perpendicular to the coating/substrate interface. In order to evaluate the effects of substrate preheating on the occurrence of micro-cracks, the HACs were deposited at different initial substrate temperatures (TS = 20, 100 and 200?C). The changes in phase composition and HA splat morphology with TS were observed and were correlated with the cracking occurrence. The results showed that severe cutting conditions introduced a localized cracking in the regions of HACs dominantly attributed to the brittle hydroxyl-deficient amorphous calcium phosphate (ACP) phase. This effect was particularly observable in the HACs deposited without preheating of substrate. On the other hand, the preheating of substrate reduced the presence of micro-cracks and caused insignificant changes in the average local phase composition. In HACs deposited with preheating of substrate, the HA splats (of which HACs are composed) were thinner and recrystallized HA regions seemed smaller in size and more evenly distributed. These results implied potentially important roles of the HA splat formation mechanism on the distribution of ACP and recrystallized HA regions in the as-plasma-sprayed HACs and the cracking resistance of HACs.

The synergic role of collagen and citrate in stabilizing amorphous calcium phosphate precursors with platy morphology.

Citrate is an important component of the bone organic matrix but its specific role in bone mineralization is presently unclear. In this work, bioinspired in vitro collagen mineralization experiments in the presence of citrate have been carried out and the combined role of collagen and citrate in controlling mineral formation has been addressed for the first time. Through X-ray scattering and Atomic Force Microscopy characterizations on mineralized and non-mineralized collagen fibrils, we have found that citrate in synergy with collagen stabilizes an amorphous calcium phosphate (ACP) phase with platy morphology over one week and controls its deposition on collagen fibrils.

Role of magnesium on the biomimetic deposition of calcium phosphate

Biomimetic depositions of calcium phosphate (CaP) are carried out using simulated body fluid (SBF), calcifying solution and newly developed magnesium containing calcifying solution. Calcium phosphate has a rich phase diagram and is well known for its excellent biocompatibility and bioactivity. The most common phase is hydroxyapatite (HAp), an integral component of human bone and tooth, widely used in orthopedic and dental applications. In addition, calcium phosphate nanoparticles show promise for the targeted drug delivery. The doping of calcium phosphate by magnesium, zinc, strontium etc. can change the protein uptake by CaP nanocrystals. This work describes the role of magnesium on the nucleation and growth of CaP on Ti and its oxide substrates. X-ray diffraction studies confirm formation of HAp nanocrystals which closely resemble the structure of bone apatite when grown using SBF and calcifying solution. It has been observed that magnesium plays crucial role in the nucleation and growth of calcium phosphate. A low magnesium level enhances the crystallinity of HAp while higher magnesium content leads to the formation of amorphous calcium phosphate (ACP) phase. Interestingly, the deposition of ACP phase is rapid when magnesium ion concentration in the solution is 40% of calcium plus magnesium ions concentration. Moreover, high magnesium content alters the morphology of CaP films.

A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix.

The modification of soft hydrogels with hard inorganic components is a method used to form composite materials with application in non-load-bearing bone tissue engineering. The inclusion of an inorganic component may provide mechanical enhancement, introduce osteoconductive or osteoinductive properties, or change other aspects of interactions between native or implanted cells and the material. A thorough understanding of the interactions between such components is needed to improve the rational design of such biomaterials. To achieve this goal, model systems which could allow study of the formation and transformation of mineral phases within a hydrogel network with a range of experimental methods and high spatial and time resolution are needed. Here, we report a detailed investigation of the formation and transformation process of calcium phosphate mineral within an alginate hydrogel matrix. A combination of optical microscopy, confocal Raman microspectroscopy and electron microscopy was used to investigate the spatial distribution, morphology and crystal phase of the calcium phosphate mineral, as well as to study transformation of the mineral phases during the hydrogel mineralization process and upon incubation in a simulated body fluid. It was found, that under the conditions used in this work, mineral initially formed as a metastable amorphous calcium phosphate phase (ACP). The ACP particles had a distinctive spherical morphology and transformed within minutes into brushite in the presence of brushite seed crystals or into octacalcium phosphate, when no seeds were present in the hydrogel matrix. Incubation of brushite-alginate composites in simulated body fluid resulted in formation of hydroxyapatite. The characterization strategy presented here allows for non-destructive, in situ observation of mineralization processes in optically transparent hydrogels with little to no sample preparation. The precipitation and transformations of calcium phosphates (CaP) is a complex process, where both formation kinetics and the stability of different mineral phases control the outcome. This situation is even more complex if CaP is precipitated in a hydrogel matrix, where one can expect the organic matrix to modulate crystallization by introducing supersaturation gradients or changing the nucleation and growth kinetics of crystals. In this study we apply a range of characterization techniques to study the mineral formation and transformations of CaP within an alginate matrix with spatiotemporal resolution. It demonstrates how a detailed investigation of the mineral precipitation and transformations can aid in the future rational design of hydrogel-based materials for bone tissue engineering and studies of biomineralization processes.

Synthesis, Characterization and in-vitro Study of Chitosan/Gelatin/Calcium Phosphate Hybrid Scaffolds Fabricated Via Ion Diffusion Mechanism for Bone Tissue Engineering

In this study, biomimetic scaffolds were designed to investigate calcium phosphate formation via a double diffusion mechanism within a gelatin/chitosan hydrogel in biological pH and temperature. Three types of samples with initial percentages of chitosan (20, 30 and 40 wt. %) were prepared. Diffusion of calcium and phosphate ions through the hydrogel formed a precipitation layer. Samples were freeze dried to form porous scaffolds and soaked in glutaraldehyde to increase their mechanical properties. X-ray diffraction (XRD), Fourier transform infra-red (FTIR) spectroscopy and scanning electron microscopy (SEM) were employed to investigate the microstructure and to characterize the prepared scaffolds. Analysis of precipitation indicated the presence of brushite and hydroxyapatite. The amorphous calcium phosphate phase converted into crystalline hydroxyapatite after immersion in simulated body fluid which mimics the formation of hydroxyapatite in the human body. FTIR results suggested the presence of structural hydroxyl and phosphate bonds in the structure of the prepared scaffolds which could be due to the formation of hydroxyapatite. With increasing amount of chitosan in the composite scaffold, the water up-take ability was increased from 380 to 660 %, yield strength and Young’s modulus slightly decreased and the crystalinity of the precipitated phase increased. Mechanical properties obtained from the samples were in the range of cancellous bone. MTT assay results and alkaline phosphatase activity showed prepared scaffolds had proper biocompatibility.

Biomimetic mineralization of synthetic collagen-like peptide: a bottom-up approach to design advanced nanocomposite scaffolds for tissue engineering

Event Abstract Back to Event Biomimetic mineralization of synthetic collagen-like peptide: a bottom-up approach to design advanced nanocomposite scaffolds for tissue engineering Gloria Belen Ramirez Rodriguez1, José Manuel Delgado-López2, Michele Iafisco1, Gopal Shankar3, Monica Montesi1, Silvia Panseri1, Monica Sandri1, Simone Sprio1, Bas Kluijtmans4 and Anna Tampieri1 1 Institute of Science and Technology for Ceramic, Bioceramics and Bio-hybrid Composites, Italy 2 Instituto Andaluz de Ciencias de la Tierra, IACT-UGR-CSIC, Spain 3 Rizzoli Orthopaedic Institute, Italy 4 FUJIFILM Manufacturing Europe B.V., TRL Life Science, Netherlands Bone extracellular matrix (ECM) consists in self-assembled nanostructure of proteins (mainly type I collagen) and poor crystalline hydroxyapatite nanocrystals (HA) that surrounds and affects mesenchymal stem cell adhesion, proliferation and differentiation through physico-chemical processes[1]. In attempts to orchestrate in vivo bone environment, we propose biomineralization of a synthetic collagen peptide (SCP, manufactured as CellnestTM by FUJIFILM Europe B.V.). Furthermore, inspired by non-stoichiometric composition of bone apatite, magnesium (Mg) is introduced during mineralization process. Mg controls HA crystallization process as well as can stimulate the osteoblast proliferation[2] and inhibit bacterial adhesion[3]. Variation on morphological and chemical features of HA related to the effect of SCP and Mg during mineralization affect surface nanotopography and consequently micro- and macroscopic interfacial scaffold properties with great relevance on cell interaction. Mineralization of synthetic collagen like-peptide (SCP) was carried out by neutralization process[4] in the absence (SCP-HA) and in the presence of magnesium (SCP-MgHA). The effect of SCP and Mg in HA crystallization was evaluated through the morphological characterization by X-Ray powder diffraction (patterns analyzed by Debye Function Analysis[5]) and transmission electron microscopy (TEM). Chemical analysis was performed by Raman spectroscopy, inductively coupled plasma optical emission spectrometry (ICP-OES) and thermogravimetric analysis. Looking ahead, mineralized solution was directly freeze-dried for the development of porous 3D scaffolds. After that, scaffolds were chemical crosslinked to ensure their stability for long-term biomedical applications. Scaffold surface topography was assessed by High-Resolution Atomic Force Microscopy (HR-AFM). The biological response of MC3T3-E1 cell line to scaffolds was assessed, too. Poor crystalline carbonated HA with similar features to that of bone apatites was synthesized. The amorphous calcium phosphate phase (ACP) was present in all conditions being the main phase in SCP-MgHA mineralization experiments which indicated the synergistic effect of SCP and Mg in stabilization of amorphous phase. Moreover, HR-AFM images of scaffold surface showed that mineralization under this condition (SCP-MgHA) lead to homogeneous mineral distribution while non-covered zones together with isolated HA nanocrystals and/or HA aggregates appeared when mineralization is carried out in the absence of Mg (SCP-HA). MC3T3-E1 cells spread inside the material and exhibited their characteristic morphology which could indicate the osteoconductive effects of designed scaffolds. Figure 1. 3D HR-AFM images of non-mineralized and mineralized scaffold surface as well as surface roughness in terms of root mean square (RMS) roughness (Rq) for scan area 2.5µm x 2.5 µm. Figure 2. SEM micrographs of MC3T3-E1 cells attached to non-mineralized and mineralized scaffolds after 7 days of culture. Herein, we propose biomineralization as a versatile route for the development of biomaterials for bone tissue engineering from nano- to macro-scopic scale. The introduction of foreign ions during mineralization allows us to modify physico-chemical properties that cue cell behavior. In our studies, synergistic effect of SCP and Mg on mineralization provided a homogeneous amorphous mineral distribution triggering to a more resorbable biomaterial. In vitro studies showed that cells attached, proliferated and colonized the scaffolds after 7 days. The research leading to these results has received funding from the European Union Seventh Framework Programme FP7-PEOPLE-2013-ITN under grant agreement n 607051

Fabrication and evaluation of Nb-containing amorphous calcium phosphate coating film on Ti

Event Abstract Back to Event Fabrication and evaluation of Nb-containing amorphous calcium phosphate coating film on Ti Kyosuke Ueda1, Takayuki Narushima1, Akiko Obata2, Toshihiro Kasuga2 and Kouetsu Ogasawara3 1 Tohoku University, Department of Materials Processing, Japan 2 Nagoya Institute of Technology, Department of Frontier Materials, Japan 3 Tohoku University, Institute of Development, Aging and Cancer, Japan Introduction: Calcium phosphate coating can potentially improve the bone compatibility of titanium (Ti). We previously reported an amorphous calcium phosphate (ACP) coating film fabricated using radiofrequency (RF) magnetron sputtering[1]. Evaluated in vivo, the ACP coating film showed high bioresorbability, and dissolution of this coating improved the bone biocompatibility of Ti[2]. Controlling the bioresorbability of the ACP coating film can extend its application to areas such as drug delivery systems (DDSs). It has been reported that niobium (Nb) stabilizes calcium phosphate glasses and decreases their resorption in simulated body fluids. Additionally, Nb ions enhance osteoblast differentiation and mineralization[3]. In this study, ACP coating films with various amounts of Nb were fabricated by RF magnetron sputtering. Subsequently, bonding strength between coating films and substrates, resorbability in simulated body fluid, and gene expression in cell culture were investigated. Materials and Methods: Coating films were fabricated by RF magnetron sputtering on mirror-polished commercially pure (CP) Ti and blasted Ti-6Al-4V substrates (10×10×1 mm) using sintered target consisting of calcium phosphate and Nb2O5. The coating films were analyzed by ICP, XRD, Raman spectroscopy, and XPS. The bonding strength between the coating film and substrate was measured by the pulling test. Amount of ions eluted from the coating film in Tris buffer solution was determined. Quantitative analysis of gene expression from SaOS-2 cells was conducted by real-time PCR. Results and Discussion: Nb content in coating films increased and Ca/P atomic ratio of coating films decreased with addition of Nb. It is speculated that Nb reached the substrate with higher probability compared with Ca and P during sputtering. Moreover, addition of Nb stabilized the ACP phase. Raman spectra of the Nb-containing ACP coating films showed that the addition of Nb caused an increase and decrease in NbO4 and (O-P-O)s(Q0) peak intensities, respectively. Thus, Nb probably acted as network former. Bonding strength between the coating film and blasted substrate was >60 MPa, which is greater than that recommended in ISO13779-2 (>15 MPa). Figure 1 shows the effect of Nb content in the coating film on amounts of eluted ions after immersion of coating films in Tris buffer solution for 86.4 ks. Total amount of eluted ions from the coating films decreased with an increase in Nb content of the coating films. It was considered that the formation of NbO4 structure was one of the reasons for suppressing ion elution. The amount of eluted Nb ion was constant, independent of Nb content in the coating films. Gene expressions such as ALP, COLI and TGF-β1 from Nb-containing ACP coating films were identical to that from non-coated substrate after a 7-day cell culture. Conclusion: Nb ions were act as network former in the ACP coating film and controlled the bioresorbability of ACP coating films. Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

X-ray diffraction investigation of amorphous calcium phosphate and hydroxyapatite under ultra-high hydrostatic pressure

The changes in the crystal structures of synthetically prepared amorphous calcium phosphate (ACP) and hydroxyapatite (HAP) in water (1:1 mass ratio) were studied by synchrotron X-ray diffraction (XRD) under ultra-high hydrostatic pressures as high as 2.34 GPa for ACP and 4 GPa for HAP. At ambient pressure, the XRD patterns of the ACP and HAP samples in capillary tubes and their environmental scanning electron micrographs indicated amorphous and crystalline characteristics for ACP and HAP, respectively. At pressures greater than 0.25 GPa, an additional broad peak was observed in the XRD pattern of the ACP phase, indicating a partial phase transition from an amorphous phase to a new high-pressure amorphous phase. The peak areas and positions of the ACP phase, as obtained through fitting of the experimental data, indicated that the ACP exhibited increased pseudo-crystalline behavior at pressures greater than 0.96 GPa. Conversely, no structural changes were observed for the HAP phase up to the highest applied pressure of 4 GPa. For HAP, a unit-cell reduction during compression was evidenced by a reduction in both refined lattice parameters a and c. Both ACP and HAP reverted to their original structures when the pressure was fully released to ambient pressure.

Nanoscale analysis of calcium phosphate films obtained by RF magnetron sputtering during the initial stages of deposition

In the present work, we investigated the nanostructure of calcium phosphate films formed during the initial stages of deposition (between 30 and 240s; deposition rate of 3.7nm/min) by radio-frequency magnetron sputtering. The sputtering deposition parameters and the plasma energy were previously adjusted to produce nearly stoichiometric nanocrystalline hydroxyapatite films after 180min. Grazing-incidence X-ray diffraction and X-ray photoelectron spectroscopy analyses revealed that the films formed in the initial stages were composed of an amorphous calcium phosphate phase with a Ca/P ratio Ca/P=1.60±0.12, which is close to that of hydroxyapatite. Using high-resolution transmission electron microscopy, we demonstrated that the films were not a unique amorphous phase. An additional phase composed of dense calcium phosphate nanospheres was found in all periods and investigated. The nanospheres grew in size as the deposition time increased, and small nanospheres were always observed. This observation suggests the continuous formation of nanospheres at the surface of the film. Nanocrystalline regions were revealed by electron diffraction and high-resolution electron microscopy after a deposition of 60s with a corresponding film thickness of 3.7nm. Electron energy-loss spectroscopy analysis comparing individual nanospheres to the amorphous calcium phosphate layer showed differences in the oxygen K edge, suggesting a difference in the chemical bonding of the oxygen species. Nanospheres were observed in association with isolated crystals, suggesting that the nanospheres may participate in the crystallization of hydroxyapatite according to a heterogeneous nucleation process.

Biomineralization of nanoscale single crystal hydroxyapatite.

The chemical and physical characteristics of nanocrystalline hydroxyapatite particles which formed during the subcutaneous implantation of crab shell in Sprague-Dawley rats were studied using selected area electron diffraction (SAED) and high resolution transmission electron microscopy (HRTEM). The initial SAED characterization evidence indicated the presence of an amorphous calcium phosphate phase. The electron dense nanophase particles which formed in the wound healing zone displayed broad diffuse rings which usually indicate a low crystalline order or amorphous phase. High resolution transmission electron microscopy (HRTEM) revealed that these mineralized regions contained discrete single crystal particles less than 5nm in size. Micrographs taken at successively higher magnifications revealed very small nanoparticles with a hexagonal arrangement of ion channels with characteristic spacing of 0.54nm and 0.23nm. This study revealed that single crystal hydroxyapatite nanoparticles consisting of only a few unit cells formed via a biomineralization directed process.

A safer disposal of hazardous phosphate coating sludge by formation of an amorphous calcium phosphate matrix

Phosphate coating hazardous wastes originated from the automotive industry were efficiently encapsulated by an acid–base reaction between phosphates present in the sludge and calcium aluminate cement, yielding very inert and stable monolithic blocks of amorphous calcium phosphate (ACP). Two different compositions of industrial sludge were characterized and loaded in ratios ranging from 10 to 50 wt.%. Setting times and compressive strengths were recorded to establish the feasibility of this method to achieve a good handling and a safe landfilling of these samples. Short solidification periods were found and leaching tests showed an excellent retention for toxic metals (Zn, Ni, Cu, Cr and Mn) and for organic matter. Retentions over 99.9% for Zn and Mn were observed even for loadings as high as 50 wt.% of the wastes. The formation of ACP phase of low porosity and high stability accounted for the effective immobilization of the hazardous components of the wastes.

Mesoscopically ordered bone-mimetic nanocomposites.

A sustainable approach that highly mimics bone-material deposition is reported to produce mechanically stable, degradable composites with nanostructures resembling that of natural bone. Molecular self-assembly combining intermolecular crosslinking leads to resilient matrices possessing long-range ordered aqueous domains, inside which moderately aligned poorly crystalline apatite is converted from the transient amorphous calcium phosphate phase.

Crystalline nano-coatings of fluorine-substituted hydroxyapatite produced by magnetron sputtering with high plasma confinement

A radio-frequency magnetron sputtering technique operating in right-angle geometry (RAMS) with high plasma confinement was revised to produce thin films (15–570nm) of fluorine-substituted hydroxyapatite, FHA, adapted to be used as nano-coatings for biomedical implants. An electron temperature of Teff≈9.0eV and a plasma electron density of 1.2×1015m−3 assured the nucleation of an amorphous fluorine-substituted hydroxyapatite phase on Si and Ti surfaces. With the aid of a Langmuir probe, the RAMS plasma energy was tuned to control the coating stoichiometry and the ratio between the crystalline and amorphous phases. The energy delivered over time from the bombardment of ions and electrons transformed the amorphous calcium phosphate phase into crystalline fluorine-substituted hydroxyapatite. The crystalline films were obtained at room temperature. The partial substitution of OH− for F− in the HA structure was confirmed by X-ray diffraction using synchrotron radiation in grazing-incidence mode, X-ray photoelectron spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy. High-resolution transmission electron microscopy carried out on cross-section film samples prepared by a focused ion beam (FIB) technique revealed that the film ultrastructure was composed of columnar crystals oriented perpendicularly to the substrate surface. The crystals were connected to the substrate surface by ordered nanolayers, indicating the existence of a continuous binding between the two materials. This work demonstrates that the RAMS technique is able to produce FHA nano-coatings with controlled chemical compositions and structures on metallic implants for clinical applications.

In Vitro Analysis of Nanoparticulate Hydroxyapatite/Chitosan Composites as Potential Drug Delivery Platforms for the Sustained Release of Antibiotics in the Treatment of Osteomyelitis

Nanoparticulate composites of hydroxyapatite (HAp) and chitosan were synthesized by ultrasound-assisted sequential precipitation and characterized for their microstructure at the atomic scale, surface charge, drug release properties, and combined antibacterial and osteogenic response. Crystallinity of HAp nanoparticles was reduced because of the interference of the surface layers of chitosan with the dissolution/reprecipitation-mediated recrystallization mechanism that conditions the transition from the as-precipitated amorphous calcium phosphate phase to the most thermodynamically stable one—HAp. Embedment of 5–10 nm sized, narrowly dispersed HAp nanoparticles within the polymeric matrix mitigated the burst release of the small molecule model drug, fluorescein, bound to HAp by physisorption, and promoted sustained-release kinetics throughout the 3 weeks of release time. The addition of chitosan to the particulate drug carrier formulation, however, reduced the antibacterial efficacy against Saureus. Excellent cell spreading and proliferation of osteoblastic MC3T3-E1 cells evidenced on microscopic conglomerates of HAp nanoparticles in vitro also markedly diminished on HAp/chitosan composites. Mitochondrial dehydrogenase activity exhibited normal values only for HAp/chitosan particle concentrations of up to 2 mg/cm2 and significantly dropped, by about 50%, at higher particle concentrations (4 and 8 mg/cm2). The gene expression of osteocalcin, a mineralization inductor, and the transcription factor Runx2 was downregulated in cells incubated in the presence of 3 mg/cm2 HAp/chitosan composite particles, whereas the expression of osteopontin, a potent mineralization inhibitor, was upregulated, further demonstrating the partially unfavorable osteoblastic cell response to the given particles. The peak in the expression of osteogenic markers paralleling the osteoblastic differentiation was also delayed most for the cell population incubated with HAp/chitosan particles. Overall, the positive effect of chitosan coating on the drug elution profile of HAp nanoparticles as carriers for the controlled delivery of antibiotics in the treatment of osteomyelitis was compensated for by the lower bacteriostatic efficiency and the comparatively unviable cell response to the composite material, especially at higher dosages.

Stabilizing amorphous calcium phosphate phase by citrate adsorption

Citrate controls nucleation by association with a precursor amorphous phase, which inhibits the surface reaction for nucleation.

Improved surface bioactivity of stainless steel substrates using osteocalcin mimetic peptide

Although stainless steel has a good biocompatibility for most clinical cases, the higher tissue response (bone bonding property) is required in orthopedic field. In this study, to improve bone-bonding ability of stainless steel substrates, a specific sequence of osteocalcin mimetic peptide is used as bioactive coating material to biochemically modify the surface of metallic samples. This sequence consists of thirteen amino acids present in the first helix of osteocalcin is synthesized in amidic form and physically adsorbed on the surface of 316LS (316 low carbon surgical grade) stainless steel substrates. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to characterize the surface of peptide coated and uncoated substrates. The bioactivity and bone bonding ability of coated and uncoated substrates are assessed by level of hydroxyapatite formation, using transmission electron microscopy (TEM), energy-dispersive x-ray (EDS), and scanning electron microscopy (SEM). The pre-osteoblast cell attachment and proliferation are also evaluated by MTT assay. The results show that the surface of coated sample is homogenously covered by the peptide and display a rougher surface relative to uncoated sample. TEM images reveal the formation of plate-like hydroxyapatite crystals in the presence of the peptide and an amorphous calcium phosphate phase without the peptide. Pre-osteoblast cells proliferation is significantly higher on the surface of peptide coated substrate, while cell attachment remains unaffected by the peptide coatings. Pre-osteoblast cells also demonstrate a higher degree of spreading on the surface of coated sample. It is believed that osteocalcin mimetic peptide improve surface bioactivity and promote hydroxyapatite crystal formation may lead to increased mineralization and bone formation on the surface of metallic biomedical devices.