Effect of Precursor Deficiency Induced Ca/P Ratio on Antibacterial and Osteoblast Adhesion Properties of Ag-Incorporated Hydroxyapatite: Reducing Ag Toxicity.

Ag-containing hydroxyapatite (HA) can reduce risks associated with bacterial infections which may eventually require additional surgical operations to retrieve a failed implant. The biological properties of HA in such applications are strongly affected by its composition in terms of dopants as well as Ca/P stoichiometry, which can be easily controlled by altering processing parameters, such as precursor concentrations. The objective of this in vitro study was to understand the effect of variations in HA precursor solutions on antibacterial properties against Escherichia coli (E. coli) and for promoting osteoblast (bone-forming cell) adhesion on Ag incorporated HA (AgHA) which has not yet been investigated. For this, two groups of AgHAs were synthesized via a precipitation method by adjusting precursor reactants with a stoichiometric value of 1.67, being either (Ca + Ag)/P (Ca-deficient) or Ca/(P + Ag) (P-deficient), and were characterized by XRD, FTIR, and SEM-EDS. Results showed that Ag+ incorporated into the Ca2+ sites was associated with a corresponding OH- vacancy. Additional incorporation of CO32- into PO43- sites occurred specifically for the P-deficient AgHAs. While antibacterial properties increased, osteoblast adhesion decreased with increasing Ag content for the Ca-deficient AgHAs, as anticipated. In contrast, significant antibacterial properties with good osteoblast behavior were observed on the P-deficient AgHAs even with a lower Ag content, owing to carbonated HA. Thus, this showed that by synthesizing AgHA using P-deficient precursors with carbonate substitution, one can keep the antibacterial properties of Ag in HA while reducing its toxic effect on osteoblasts.

In vitro performance of Ag-incorporated hydroxyapatite and its adhesive porous coatings deposited by electrostatic spraying.

Bacterial infection of implanted materials is a significant complication that might require additional surgical operations for implant retrieval. As an antibacterial biomaterial, Ag-containing hydroxyapatite (HA) may be a solution to reduce the incidences of implant associated infections. In this study, pure, 0.2mol% and 0.3mol% Ag incorporated HA powders were synthesized via a precipitation method. Colloidal precursor dispersions prepared from these powders were used to deposit porous coatings onto titanium and stainless steel substrates via electrostatic spraying. The porous coating layers obtained with various deposition times and heat treatment conditions were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Scratch tests were conducted to assess the adhesion strength of the coating. Antibacterial activity of Ag-incorporated HA was tested towards Escherichia coli (E. coli) at various incubation times. Osteoblast adhesion on Ag-incorporated HA was evaluated to assess biocompatibility. Improvement in adhesion strength of the coating layer was observed after the heat treatment process due to mutual ionic diffusion at the interface. The Ag-incorporated HA killed all viable E. coli after 24h of incubation, whereas no antibacterial activity was detected with pure HA. In addition, in vitro cell culture tests demonstrated osteoblast adhesion similar to pure HA, which indicated good cytocompatibility. In summary, results of this study provided significant promise for the future study of Ag-incorporated HA for numerous medical applications.

Synthesis and characterization of Ag-containing calcium phosphates with various Ca/P ratios.

Ag-containing calcium phosphate (CaP) powders were synthesized by a precipitation method using aqueous solutions of calcium nitrate, silver nitrate, and ammonium phosphate. The powders were sintered at temperatures ranging from 1173 to 1473 K. The charged atomic ratios of (Ca+Ag)/P and Ag/(Ca+Ag) in solution were varied from 1.33 to 1.67 and from 0 to 0.30, respectively. The Ag content in the as-precipitated CaP powders increased with the charged Ag/(Ca+Ag) atomic ratio in solution and was lower than the charged Ag/(Ca+Ag) value. The as-precipitated CaP powders consisted of hydroxyapatite (HA) as the main phase. Ag nanoparticles were observed on the as-precipitated HA particles under all conditions of Ag addition. After the sintering, HA, ß-TCP (tricalcium phosphate), α-TCP, and ß-CPP (calcium pyrophosphate) were mainly detected as CaPs on the basis of the Ca/P atomic ratio of the as-precipitated powders. The addition of Ag stabilized the ß-TCP phase, and the distribution of Ag in ß-TCP was homogeneous. A metallic Ag phase coexisted with HA. The solubility of Ag in HA was estimated to be 0.0019-0.0061 (Ag/(Ca+Ag)) atomic ratio, which was lower than that in ß-TCP (higher than 0.0536) and higher than that of ß-CPP (below the detection limit of analyses).

The effect of combined delivery of recombinant human bone morphogenetic protein-2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium-phosphate-coated implants on osseointegration.

OBJECTIVES: The delivery of growth factors for enhanced osseointegration depends on the effectiveness of the carrier systems at the bone-implant interface. This study evaluated the effect of solo and dual delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF(165) ) from biomimetically octacalcium phosphate-coated implants on osseointegration. MATERIALS AND METHODS: Biomimetic implants, bearing either a single growth factor (BMP or VEGF) or their combination (BMP+VEGF), were established, and compared with acid-etched (AE, control) and biomimetic implants without growth factor (CAP). Implants were placed into frontal skulls of nine domestic pigs. The quality of osseointegration was evaluated using microradiographic and histomorphometric analysis of bone formation inside four defined bone chambers of the experimental implant at 1, 2 and 4 weeks. RESULTS: Biomimetic implants, either with or without growth factor, showed enhanced bone volume density (BVD) values after 2 and 4 weeks. This enhancement was significant for the BMP and BMP+VEGF group compared with the control AE group after 2 weeks (P<0.05). All biomimetic calcium-phosphate (Ca-P) coatings exhibited significantly enhanced bone-implant contact (BIC) rates compared with the uncoated control surface after 2 weeks (P<0.05). However, the combined delivery of BMP-2 and VEGF did not significantly enhance BIC at the final observation period. CONCLUSION: It was concluded that the combined delivery of BMP-2 and VEGF enhances BVD around implants, but not BIC. Therefore, it may be assumed that changes in the surface characteristics should be considered when designing growth factor-delivering surfaces.

Osteoblast adhesion on novel machinable calcium phosphate/lanthanum phosphate composites for orthopedic applications.

Lanthanum phosphate (LaPO(4), LP) was combined with either hydroxyapatite (HA) or tricalcium phosphate (TCP) to form novel composites for orthopedic applications. In this study, these composites were prepared by wet chemistry synthesis and subsequent powder mixing. These HA/LP and TCP/LP composites were characterized in terms of phase stability and microstructure evolution during sintering using X-ray diffraction (XRD) and scanning electron microscopy (SEM). Their machinability was evaluated using a direct drilling test. For HA/LP composites, LP reacted with HA during sintering and formed a new phase, Ca(8)La(2)(PO(4))(6)O(2), as a reaction by-product. However, TCP/LP composites showed phase stability and the formation of a weak interface between TCP and LP machinability when sintered at 1100 degrees C, which is crucial for achieving desirable properties. Thus, these novel TCP/LP composites fulfilled the requirements for machinability, a key consideration for manufacturing orthopedic implants. Moreover, the biocompatibility of these novel LP composites was studied, for the first time, in this paper. In vitro cell culture tests demonstrated that the LP and its composites supported osteoblast (bone-forming cell) adhesion similar to natural bioceramics (such as HA and TCP). In conclusion, these novel LP composites should be further studied and developed for more effectively treating bone related diseases or injuries.

An in vitro evaluation of the Ca/P ratio for the cytocompatibility of nano-to-micron particulate calcium phosphates for bone regeneration.

Calcium phosphate based bioceramics have been widely used for orthopedic applications due to their chemical similarity to natural bone. The Ca/P stoichiometry of calcium phosphates strongly influences their performance under biological conditions, which have not yet been fully elucidated to date. For this reason, the objective of this in vitro study was to understand the relationship between the Ca/P ratio of nano-to-micron particulate calcium phosphate substrates and their biological properties, such as osteoblast (bone-forming cell) viability, collagen production, alkaline phosphatase activity and nitric oxide (NO) production. A group of calcium phosphates with Ca/P ratios between 0.5 and 2.5 were obtained by intentionally adjusting the Ca/P stoichiometry of the initial reactants necessary for calcium phosphate precipitation. For samples with 0.5 and 0.75 Ca/P ratios, tricalcium phosphate (TCP) and Ca(2)P(2)O(7) phases were observed. In contrast, for samples with 1.0 and 1.33 Ca/P ratios, the only stable phase was TCP. For samples with a 1.5 Ca/P ratio, the TCP phase was dominant; however, small amounts of the hydroxyapatite (HA) phase started to appear. For samples with a 1.6 Ca/P ratio, the HA phase was dominant. Lastly, for samples with 2.0 and 2.5 Ca/P ratios, the CaO phase started to appear in the HA phase which was the dominant phase. Moreover, the average grain size and the average pore size decreased from micron-scale (e.g. 1370nm for a 0.5 Ca/P ratio) to nano-scale (e.g. 262nm for a 2.5 Ca/P ratio) with increasing Ca/P ratios. The porosity (%) of calcium phosphate substrates also decreased with increasing Ca/P ratios. Previous in vitro results demonstrated increased osteoblast adhesion on calcium phosphates with higher Ca/P ratios (up to 2.5). The present study showed that the collagen production by osteoblasts was similar between all the calcium phosphates but slightly lower with a 1.6 Ca/P ratio. Greater alkaline phosphatase activity by osteoblasts was observed in all the cultures with various calcium phosphates (0.5-2.5 Ca/P ratios) than in the control (only cells in culture). Ca/P ratios of <2 and 1 optimized osteoblast viability and promoted alkaline phosphatase activity in osteoblasts, respectively. However, the presence of the CaO phase in Ca/P ratios 2.0 increased osteoblast NO production and decreased osteoblast viability. In summary, this study provided evidence that the Ca/P ratio of calcium phosphate is a very important factor that should be considered when selecting nano-to-micron particulate calcium phosphates for various orthopedic applications.

Increased osteoblast adhesion on nanoparticulate calcium phosphates with higher Ca/P ratios.

The biological properties of calcium phosphate-derived materials are strongly influenced by changes in Ca/P stoichiometry and grain size, which have not yet been fully elucidated to date. For this reason, the objective of this in vitro study was to understand osteoblast (bone forming cells) adhesion on nanoparticulate calcium phosphates of various Ca/P ratios. A group of calcium phosphates with Ca/P ratios between 0.5 and 2.5 were obtained by adjusting the Ca/P stoichiometry of the initial reactants necessary for calcium phosphate precipitation. For samples with 0.5 and 0.75 Ca/P ratios, tricalcium phosphate (TCP) and Ca(2)P(2)O(7) phases were observed. In contrast, for samples with 1.0 and 1.33 Ca/P ratios, the only stable phase was TCP. For samples with 1.5 Ca/P ratios, the TCP phase was dominant, however, small amounts of the hydroxyapatite (HA) phase started to appear. For samples with 1.6 Ca/P ratios, the HA phase was dominant. Last, for samples with 2.0 and 2.5 Ca/P ratios, the CaO phase started to appear in the HA phase, which was the dominant phase. Moreover, the average nanometer grain size, porosity (%), and the average pore size decreased in general with increasing Ca/P ratios. Most importantly, results demonstrated increased osteoblast adhesion on calcium phosphates with higher Ca/P ratios (up to 2.5). In this manner, this study provided evidence that Ca/P ratios should be maximized (up to 2.5) in nanoparticulate calcium phosphate formulations to increase osteoblast adhesion, a necessary step for subsequent osteoblast functions such as new bone deposition.

Increased osteoblast adhesion on nanograined hydroxyapatite and tricalcium phosphate containing calcium titanate.

Depending on the coating method utilized and subsequent heat treatments (such as through the use of plasma-spray deposition), inter-diffusion of atomic species across titanium (Ti) and hydroxyapatite (HA) coatings may result. These events may lead to structural and compositional changes that consequently cause unanticipated HA phase transformations which may clearly influence the performance of an orthopedic implant. Thus, the objective of the present in vitro study was to compare the cytocompatibility properties of chemistries that may form at the Ti:HA interface, specifically HA, tricalcium phosphate (TCP), HA doped with Ti, and those containing calcium titanate (CaTiO(3)). In doing so, results of this study showed that osteoblast (bone-forming cells) adhesion increased with greater CaTiO(3) substitutions in either HA or TCP. Specifically, osteoblast adhesion on HA and TCP composites with CaTiO(3) was almost 4.5 times higher than that over pure HA. Material characterization studies revealed that enhanced osteoblast adhesion on these compacts may be due to increasing shrinkage in the unit lattice parameters and decreasing grain size. Although all CaTiO(3) composites exhibited excellent osteoblast adhesion results, Ca(9)HPO(4)(PO(4))(5)OH phase transformation into TCP/CaTiO(3) increased osteoblast adhesion the most; because of these reasons, these materials should be further studied for orthopedic applications.

Increased osteoblast adhesion on titanium-coated hydroxylapatite that forms CaTiO3.

CaTiO(3) is a strong candidate to form at the interface between hydroxylapatite (HA) and titanium implants during many coating procedures. However, few studies have compared the cytocompatibility properties of CaTiO(3) to HA pertinent for bone-cell function. For this reason, the objective of the present in vitro study was to determine the ability of bone-forming cells (osteoblasts) to adhere on titanium coated with HA that resulted in the formation of CaTiO(3). To accomplish the formation of CaTiO(3), titanium was coated on HA discs and annealed either under air or a N(2)+H(2) environment. Materials were characterized by X-ray diffraction (XRD), Rutherford backscattering spectroscopy (RBS), and atomic force microscopy (AFM). These characterization techniques demonstrated the formation of a nanometer rough CaTiO(3) layer as a consequence of interactions between HA and titanium during coating conditions. Results from cytocompatibility tests revealed increased osteoblast adhesion on materials that contained CaTiO(3) compared to both pure HA and uncoated titanium. The greatest osteoblast adhesion was observed on titanium-coated HA annealed under air conditions. Because adhesion is a crucial prerequisite to subsequent functions of osteoblasts (such as the deposition of calcium containing mineral), the present in vitro results imply that orthopedic coatings that form CaTiO(3) could increase osseointegration with juxtaposed bone needed for increased implant efficacy.

Hydroxylapatite and titanium: interfacial reactions.

The chemical reactions between hydroxylapatite (HA) and titanium were studied in three different kinds of experiments to increase understanding of how to bond HA to titanium for implant materials. HA powder was bonded to a titanium rod with hot isostatic pressing. Interdiffusion of the HA elements and titanium was found in concentration profiles measured in the electron microprobe. Titanium was vapor-deposited on sintered HA discs and heated in air; perovskite (CaTiO(3)) was found on the HA surface with Rutherford backscattering and X-ray diffraction measurements. Powder composites of HA and titanium and TiO(2) were sintered at 1100 degrees C; again, perovskite was a reaction product, as well as beta-Ca(3)(PO(4))(2), from decomposition of the HA. These results demonstrate chemical reactions and interdiffusion between HA and TiO(2) during sintering, resulting in chemical bonding between HA and titanium. Thus, cracks and weakness at HA-titanium interfaces probably result from mismatch between the coefficients of thermal expansion of these materials. HA composites with other ceramics and different alloys should lead to better thermal matching and better bonding at the interface.

Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. I. Structure and microstructure.

Hydroxylapatite (HA) was made containing magnesium, zinc, cadmium, and yttrium. Salts of these cations were added to precipitating HA; the precipitates were dried and sintered at 1100 degrees C for 1 h. Lattice parameters from X-ray diffraction spectra showed that these elements were incorporated into the apatite structure at a level of 2% added fraction of calcium in HA and up to 7% for yttrium. The densities of different substituted apatites were close to theoretical for pressed and sintered samples, which is evidence for low bulk porosity. The grain sizes of substituted apatites were smaller than those of pure HA except for cadmium-containing apatite. Surfaces of etched samples showed no second phases, whereas surfaces of unetched samples showed second phases and higher porosity than etched surfaces.

Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion.

The present in vitro study investigated osteoblast adhesion on hydroxylapatite (HA) doped with either cadmium (Cd), zinc (Zn), magnesium (Mg), or yttrium (Y). Compared with any other dopant tested in the present study, osteoblast adhesion was significantly (p < 0.05) greater on HA doped with Y after 4 h; in addition, osteoblast adhesion increased with concentration (2-7 mol%) of Y in HA. The findings that HA doped with greater amounts of Y adsorbed higher concentrations of calcium and, subsequently, of vitronectin and collagen (proteins known to mediate osteoblast adhesion), but not of albumin, laminin, and fibronectin, may explain the observed enhanced adhesion of osteoblasts on these substrates. Interactions (i.e., adsorption and configuration/bioactivity) of vitronectin and collagen may have been promoted by increased porosity of doped HA. Through doping with Y, the present study provided the first evidence that HA can be synthesized and processed with improved cytocompatibility properties for osteoblast adhesion, and thus offered essential information for the design of novel proactive bioceramics. Proactive bioceramics which elicit specific, timely, and desired responses from surrounding cells and tissues are necessary for improving bonding of orthopaedic/dental implants to juxtaposed bone; such osseointegration will, undoubtedly, enhance implant efficacy.