Calcium Phosphate-based Materials Research Articles

Evaluation of a β‐Calcium Metaphosphate Bone Graft Containing Bone Morphogenetic Protein‐7 in Rabbit Maxillary Defects

Calcium phosphate-based materials have been widely used as bone substitutes and more recently are being exploited together with growth factors as bone tissue engineering scaffolds regulating cell behavior. The aim of this study is to evaluate the in vitro and in vivo response to a newly developed calcium metaphosphate (CMP) bone graft, with and without bone-stimulating growth factor. Porous scaffolds of CMP were developed and extensively tested in vitro. Subsequently, CMP grafts with osteogenic protein-1 (OP-1) (test) and without OP-1 (control) were implanted into experimental rabbit maxillary bone defects. Animals were sacrificed at 2, 4, and 8 weeks, and samples were examined with microcomputed tomography (micro-CT) and processed for histomorphometric analysis. At 8 weeks, the scaffolds containing OP-1 induced greater bone formation (P = 0.018) than CMP alone, based on histomorphometric evaluation (percentage bone area: test: 57.1 ± 5.6; control: 49.4 ± 7.7) and micro-CT analysis (percentage bone volume density: test: 63.46 ± 5.61; control: 51.20 ± 6.71). Thus, these data indicated that both test and control CMP grafts showed a good degree of bone formation. Furthermore, the CMP materials showed signs of resorption from 4 weeks, and no graft materials were observed at 8 weeks. In vitro, the OP-1 loaded graft demonstrated a release profile and bioactivity over a 28-day period. In vivo testing confirmed enhanced bone formation of the OP-1 loaded graft after 8 weeks of healing.

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer: Preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-l-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of β-tricalcium phosphate (β-TCP) bioceramics by soaking β-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of β-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the β-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of β-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.

Bioinformatics-based selection of a model cell type for in vitro biomaterial testing

Biomaterial properties can be tailored for specific applications via systematic and high-throughput screening of biomaterial–cell interactions. However, progress in material development is often hampered by the lack of adequate in vitro testing methods, frequently due to incomplete understanding of involved in vivo mechanisms. In line with drug discovery in pharmacology, a crucial step in assay development for biomaterial screening is the identification of a target to direct the screen against. Herein, the cell type to be used for screening is of essential importance and has to be carefully chosen. So far, few attention has been put on selecting a cell type specifically suitable for in vitro testing of materials for predefined applications. In this manuscript, we describe an approach to define a suitable cell type for screening assays, for which biomaterials for bone regeneration served as example. Using a bioinformatics methodology, different cell lines are compared on three well-characterized model materials. The transcriptional profiles of MG63, iMSC, SV-HFO, hPPCT, hBPCT and SW480 cells are assessed on 3 calcium phosphate-based materials to evaluate their potential application in a screening assay. We show that MG63 is the most suitable cell line to evaluate biomaterials for bone regeneration applications, evidenced by their robust gene expression differences between the 3 model materials. The gene expression differences between the cell lines were assessed based on the overall gene expression profiles and specific subsets of genes and pathways related to osteogenesis and bone homeostasis in response to the 3 materials tested. In the next phase, this cell line will be used to identify a target correlating with in vivo biomaterial performance and henceforth to develop an in vitro screening system.

Calcium phosphate-based materials of natural origin showing photocatalytic activity

Calcium phosphate based materials of natural origin with photocatalytic properties were produced. Bones of Atlantic cod fish were treated in appropriate solutions (either Ca- or Ti-containing salts) and successively annealed. Results showed multiphasic materials (hydroxyapatite, β-tricalcium phosphate and anatase titania) with excellent photocatalytic performance under both UV and visible light, with an anatase concentration of only about 2 mol%. Results with such a low amount of anatase have never been reported before for a calcium phosphate-based material; this is attributed to the presence of anatase being mainly on the surface. Single-phase hydroxyapatite (Ca10(PO4)6(OH)2 or HAp) also showed some photocatalytic properties and antibacterial activity.

Synthesis and Structural Characterization of Hydroxyapatite-Wollastonite Biocomposites, Produced by an Alternative Sol-Gel Route

Hydroxyapatite is a type of calcium phosphate-based material with great interest for biomedical applications, due to the chemical similarity between this material and the mineral part of human bone. However, synthetic hydroxyapatite is essentially brittle; the practice indicates that the use of hydroxyapatite without additives for implant production is not efficient, due to its low strength parameters. In the present work, biocomposites of hydroxyapatite-wollastonite were synthesized by an alternative sol-gel route, using calcium nitrate and ammonium phosphate as precursors of hydroxyapatite, and high purity natural wollastonite was added in ratios of 20, 50 and 80 percent by weight immersed in aqueous medium. Formation of hydroxyapatite occurs at a relatively low temperature of about 350?C, while the wollastonite remains unreacted. After that, these biocomposites were sintered at 1200?C for 5 h to produce dense materials. The characterization techniques demonstrated the presence of hydroxyapatite and wollastonite as unique phases in all products.

Differentiation of osteoblast and osteoclast precursors on pure and silicon-substituted synthesized hydroxyapatites

Calcium phosphate-based materials should show excellent bone-bonding and cell-mediated resorption characteristics at the same time, in order to be employed for bone replacement. In this perspective, pure (HAp) and silicon-substituted hydroxyapatite (Si-HAp, 1.4% wt) porous cylinders were prepared starting from synthesized powders and polyethylene spheres used as porogens, and investigated as supports for osteoblast and osteoclast progenitor differentiation. A systematic and detailed biological characterization is reported, in terms of cell adhesion, viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell in vitro behavior, comprehensive of both the osteogenesis and the bone resorption processes. In order to achieve this purpose, cytocompatibility, differentiation and gene expression by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were carried out using parietal bone-derived pre-osteoblasts obtained from neonatal mice and the bioresorption capability was assessed by seeding human peripheral blood monocytes, as osteoclast precursors. It resulted that both pure and Si-substituted HAps were able to promote differentiation of precursor cells in mature osteoblasts and osteoclasts. In particular, the Si-HAps enhanced the pre-osteoblast proliferation and showed higher osteoclast-mediated bioresorption capability, as supported by the presence of larger and more numerous resorption lacunae, whereas HAps promoted a more robust cell differentiation in terms of both osteocalcin gene expression by qRT-PCR and cell morphological evaluation by SEM analysis.

Osteoblast-like cellular response to dynamic changes in the ionic extracellular environment produced by calcium-deficient hydroxyapatite

Solution-mediated reactions due to ionic substitutions are increasingly explored as a strategy to improve the biological performance of calcium phosphate-based materials. Yet, cellular response to well-defined dynamic changes of the ionic extracellular environment has so far not been carefully studied in a biomaterials context. In this work, we present kinetic data on how osteoblast-like SAOS-2 cellular activity and calcium-deficient hydroxyapatite (CDHA) influenced extracellular pH as well as extracellular concentrations of calcium and phosphate in standard in vitro conditions. Since cells were grown on membranes permeable to ions and proteins, they could share the same aqueous environment with CDHA, but still be physically separated from the material. In such culture conditions, it was observed that gradual material-induced adsorption of calcium and phosphate from the medium had only minor influence on cellular proliferation and alkaline phosphatase activity, but that competition for calcium and phosphate between cells and the biomaterial delayed and reduced significantly the cellular capacity to deposit calcium in the extracellular matrix. The presented work thus gives insights into how and to what extent solution-mediated reactions can influence cellular response, and this will be necessary to take into account when interpreting CDHA performance both in vitro and in vivo.

Effect of strontium-substituted biphasic calcium phosphate on inflammatory mediators production by human monocytes

Calcium phosphate materials are widely used as bone substitutes because of their properties close to those of the mineral phase of bones. Nevertheless, after several months, calcium phosphate-based materials release particles that may be phagocytosed by monocytes, leading to an inflammatory reaction. Strontium is well known to counteract the osteoporosis process, but little is known about its effect on inflammatory processes. The purpose of this work was to study the effect of biphasic calcium phosphate (BCP) particles substituted with strontium on the inflammatory reaction. Human primary monocytes stimulated or not by lipopolysaccharide (LPS) were exposed to BCP particles containing strontium for 6 and 24h. Inflammatory mediators (cytokines and matrix metalloproteinases (MMPs)) production was then quantified by ELISA and zymography. We observed that the presence of strontium had few effects on unstimulated cells, but it decreased the production of pro-inflammatory cytokines and the chemokine interleukin 8 in LPS-stimulated cell-conditioned medium. This work suggests for the first time that strontium may be involved in the control of inflammatory processes following BCP phagocytosis by human monocytes.

Trends in development of calcium phosphate-based ceramic and composite materials for medical applications: Transition to nanoscale

Current status and lines of research were considered for calcium phosphate-based ceramic materials intended for application in new technologies for damaged bone tissue regeneration. The main trend is toward mimicking the composition, structure, and properties of biological bone tissues by ceramic materials. This necessitated developmental efforts on synthesizing polysubstituted, in particular nanocrystalline, biphasic calcium phosphates and on preparing ceramics and composite materials thereof. The progress achieved in this area was reviewed, and certain benefits associated with medical application of these materials were discussed.

Surface Characteristics of Nanocrystalline Apatites: Effect of Mg Surface Enrichment on Morphology, Surface Hydration Species, and Cationic Environments

The incorporation of foreign ions, such as Mg2+, exhibiting a biological activity for bone regeneration is presently considered as a promising route for increasing the bioactivity of bone-engineering scaffolds. In this work, the morphology, structure, and surface hydration of biomimetic nanocrystalline apatites were investigated before and after surface exchange with such Mg2+ ions, by combining chemical alterations (ion exchange, H2O-D2O exchanges) and physical examinations (Fourier transform infrared spectroscopy (FTIR) and high-resolution transmission electron microscopy (HRTEM)). HRTEM data suggested that the Mg2+/Ca2+ exchange process did not affect the morphology and surface topology of the apatite nanocrystals significantly, while a new phase, likely a hydrated calcium and/or magnesium phosphate, was formed in small amount for high Mg concentrations. Near-infrared (NIR) and medium-infrared (MIR) spectroscopies indicated that the samples enriched with Mg2+ were found to retain more water at their surface than the Mg-free sample, both at the level of H2O coordinated to cations and adsorbed in the form of multilayers. Additionally, the H-bonding network in defective subsurface layers was also noticeably modified, indicating that the Mg2+/Ca2+ exchange involved was not limited to the surface. This work is intended to widen the present knowledge on Mg-enriched calcium phosphate-based bioactive materials intended for bone repair applications.

In vitro biomimetic deposition of apatite on alkaline and heat treated Ti6A14V alloy surface

Titanium alloy (Ti6A14V) substrates, having the ability of biomimetic calcium phosphate-based materials, especially hydroxyapatite deposition in a simulated body fluid (SBF) means of chemical treatment (alkaline treatment) and subsequent heat treatment, was studied. The effects of alkaline treatment time, concentration and heat treatment temperature on the formation of calcium phosphate (carbonate-hydroxyapatite) on Ti6A14V surface were examined. For this purpose, the metallic substrates were treated in 0, 5 and 10 M NaOH solutions at a temperature of 60 or 80°C for 1 and 3 days. Subsequently the substrate was heat-treated at 500, 600 and 700°C for 1 h for consolidation of the sodium titanate hydrogel layer. Finally, they were soaked in SBF for 1 and 3 days. The substrate surfaces were characterized by the techniques commonly used for bulk material such as scanning electron microscopy (SEM) and thin film X-ray diffraction (TF-XRD). With regard to the SEM and TF-XRD results, the optimum process consists of 3 days soaking in 5 M NaOH in 80°C and subsequent heat treatment at 600°C for 1h. It is worth mentioning that the results showed that the apatite formed within 3 days on the specimen surfaces, however, there was no sign of apatite formation in the control samples (without alkaline and heat treatment) which was treated for up to 3 days immersion in SBF.

Influence of TiO2 and Ag2O addition on tricalcium phosphate ceramics

Degradation of implanted ceramics allows for bone in-growth and eventual replacement with natural tissue. Calcium phosphate-based materials have gained the most significant attention because of their excellent biocompatibility and compositional similarities to natural bone. Adding various dopants to these ceramics significantly influences critical properties. In this study, tricalcium phosphate (TCP) compacts were fabricated via uniaxial compression with four compositions: (i) pure TCP, (ii) TCP with 1.0 wt % TiO(2), (iii) TCP with 0.5 wt % Ag(2)O, and (iv) ternary of TCP and 1.0 wt % TiO(2), and 0.5 wt % Ag(2)O. These compacts were sintered at 1250 degrees C for 4 h to obtain dense ceramic structures. Phase analyses were carried out using an X-ray diffractometer. The presence of TiO(2) in TCP improved densification and increased compression strength from 70 (+/-25) to 145 (+/-40) MPa. The ternary composition had the highest density and compression strength of 180 (+/-15) MPa. Human osteoblast cell growth behavior was studied using an osteoprecursor cell line (OPC 1) to assure that the biocompatibility of these ceramics was not altered due to the dopants. For long-term biodegradation studies, density, weight change, surface microstructure, and uniaxial compression strength were measured as a function of time in a simulated body fluid (SBF). Weight gain in SBF correlated strongly with precipitation viewed in the inter-connected pores of the samples. After 3 months in SBF, a 35% drop in compression strength was noticed for pure TCP, but for doped compositions, no strength loss was noticed.

Functionalized Calcium Phosphate-based Nanoparticles for the Treatment of Osteoporosis

AbstractIn an effort to decrease the number of problems associated with osteoporosis, the long-term goal of the present study is to design calcium phosphate-based nanoparticles that specifically attach to areas of low bone density and once attached, allow for the targeted release of bioactive agents that can quickly increase bone formation. Efforts are focused on nanoparticles of calcium phosphate-based materials since they are similar in size and chemistry to the major inorganic components of bone. As a first step in this research, the objective of the present study was to synthesize nanoparticles of crystalline hydroxyapatite (or HA) and amorphous calcium phosphate. Crystalline HA is stable under physiological fluids and, thus, will release embedded bioactive agents slowly. Alternatively, amorphous calcium phosphate is highly biodegradable and will, thus, release embedded bioactive agents quickly. A further objective of the present study was to functionalize such inorganic biodegradable materials with amino groups which would allow for the subsequent attachment of entities to direct such nanoparticles to osteoporotic bone and increased bone formation once attached. One promising approach to direct the nanoparticles to osteoporotic bone is to attach antibodies to pentosidine on the nanoparticles since pentosidine is present in higher amounts in osteoporotic compared to healthy bone. A promising approach to increase bone growth once nanoparticles attach to osteoporotic bone, is to embed nanoparticles with regions of the bone growth factor: bone morphogenic protein-2 (or BMP-2). Results of this study demonstrated the successful synthesis of both crystalline HA and amorphous calcium phosphate nanoparticles. Furthermore, results showed that these nanoparticles can be functionalized with versatile amino groups. In this manner, this study takes the first steps toward utilizing calcium phosphate based nanoparticles to reverse bone loss associated with osteoporosis.

Analysis of the physical characterization and the tabletability of calcium phosphate-based materials

The rheological properties of the new excipients A-, Di- and Tri-tab® were in general better than those of Emcompress®, whereas a mixture of Emcompress® with magnesium stearate exhibited the best flow characteristics and the lowest coefficient of tablet weight variation. Granulometry of the xcipients demonstrates that Tri-tab® has a maximum mean diameter and a wide particle size distribution, while Emcompress® has a particle size distribution similar to that of Di-tab®. Different compression properties, namely, net work, compactibility, plasticity and ejection work were evaluated after adding a hydrophobic lubricant to the wall of the die. The compressional properties demonstrated the values of the friction parameters to be insufficient in the cae of Di- and Tri-tab® powders, therfore, lubrication would be a major factor in tableting with these excipients for direct compression. Absolutely different compressional behavior of Di-tabl was observed. Moreover, the parameters obtained from the Heckel in die tablet method were calculated in order to establish the comparative consolidation mechanisms in the excipients under study. Emcompress® showed a lower extent of brittle fracture and greater plasticity than the other calcium phosphate-based excipients.

Experimental Hydroxyapatite Cement Cranioplasty

Hydroxyapatite cement is a calcium phosphate-based material that when mixed with water forms a dense paste that sets within 15 minutes and isothermically converts in vivo to a microporous hydroxyapatite implant. This cement was used to reconstruct bilateral 2.5-cm-diameter full-thickness critical-sized parietal skull defects in six cats. One side was reconstructed with 100 percent hydroxyapatite cement, and the other with a mixture of 50 percent hydroxyapatite cement and 50 percent ground autogenous bone by weight. These animals were sacrificed at 6 and 12 months after implantation. Positive and negative controls also were prepared. The anatomic contour of the soft tissue overlying all hydroxyapatite cement implants was well maintained, there were no wound infections or structural failures, and the implants were well tolerated histologically. None of the negative (unreconstructed) control defects was completely filled with repair bone, and all positive (methyl methacrylate) controls demonstrated foreign-body giant-cell formation and fibrous encapsulation of the implants. Examination of decalcified and undecalcified sections revealed progressive but variable replacement of the cement by new bone and soft tissue without a change in the shape or volume of the hydroxyapatite cement-reconstructed areas. New bone comprised 77.3 and 64.7 percent of the tissue replacing the hydroxyapatite cement and hydroxyapatite cement-bone implants, respectively. Replacement of the hydroxyapatite cement implants by new bone is postulated to occur by a combination of osteoconduction and implant resorption. These results indicate that further experimental research leading to the possible application of hydroxyapatite cement for full-thickness calvarial defect reconstruction in humans is warranted.