Synthesis of calcium-deficient hydroxyapatite nanowires and nanotubes performed by template-assisted electrodeposition.

Hydroxyapatite (HA) has received much interest for being used as bone substitutes because of its similarity with bioapatites. In form of nanowires or nanotubes, HA would offer more advantages such as better biological and mechanical properties than conventional particles (spherical). To date, no study had allowed the isolated nanowires production with simultaneously well-controlled morphology and size, narrow size distribution and high aspect ratio (length on diameter ratio). So, it is impossible to determine exactly the real impact of particles' size and aspect ratio on healing responses of bone substitutes and characteristics of these ones; their biological and mechanical effects can never be reproducible. By the template-assisted pulsed electrodeposition method, we have for the first time succeeded to obtain such calcium-deficient hydroxyapatite (CDHA) particles in aqueous baths with hydrogen peroxide by both applying pulsed current density and pulsed potential in cathodic electrodeposition. After determining the best conditions for CDHA synthesis on gold substrate in thin films by X-ray diffraction (XRD) and Energy dispersive X-ray spectroscopy (EDX), we have transferred those conditions to the nanowires and nanotubes synthesis with high aspect ratio going until 71 and 25 respectively. Polycrystalline CDHA nanowires and nanotubes were characterized by Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). At the same time, this study enabled to understand the mechanism of nanopores filling in gold covered polycarbonate membrane: here a preferential nucleation on gold in membranes with 100 and 200 nm nanopores diameters forming nanowires whereas a preferential and randomly nucleation on nanopores walls in membranes with 400 nm nanopores diameter forming nanotubes.

Bioactivity of Biphasic Calcium Phosphate Granules, the Control of a Needle-Like Apatite Layer Formation for Further Medical Device Developments.

Biphasic calcium phosphate (BCP) bioceramics (hydroxyapatite/tricalcium phosphate, or HA/TCP) for tissue engineering and drug delivery systems is a unique know-how. A mechanical mixture of HA and TCP does not lead to such bioactive ceramics. The wet elaboration conditions of calcium-deficient apatite (CDA) or CDHA, followed by sintering, converts it into TCP and HA. The dissolution precipitation of nano-sized needle-like crystals at the surface of BCP occurs on time at body temperature. Combining several technics of characterization [scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive x-ray spectroscopy (EDX), Brunauer-Emmett-Teller method (BET), chemical analysis, x-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR)], we demonstrated an evolution on time of the HA/ß-TCP. The current paper describes the crystallographic evolution of initial ß-TCP rhombohedral crystallographic structure to microsized needle-like layer corresponding to apatitic TCP form. This phenomenon leads to an increase of the HA/TCP ratio, since hexagonal apatitic TCP is similar to hexagonal HA. However, the Ca/P ratio (reflecting the chemical composition HA/TCP) remains unchanged. Thus, the high reactivity of BCP involves dynamic evolution from rhombohedral to hexagonal structure, but not a chemical change. The dynamic process is reversible by calcination. These events are absolutely necessary for smart scaffolds in bone regeneration and orthobiology.

A Cellulose/Laponite Interpenetrated Polymer Network (IPN) Hydrogel: Controllable Double-Network Structure with High Modulus.

Laponite XLS™, which is a synthetic clay of nanometric dimensions containing a peptizing agent, has been associated with silanized hydroxypropylmethylcellulose (Si-HPMC) to form, after crosslinking, a novel composite hydrogel. Different protocols of sample preparation were used, leading to different morphologies. A key result was that the storage modulus of Si-HPMC/XLS composite hydrogel could be increased ten times when compared to that of pure Si-HPMC hydrogel using 2 wt % of Laponite. The viscoelastic properties of the composite formulations indicated that chemical and physical network structures co-existed in the Si-HPMC/XLS composite hydrogel. Images that were obtained from confocal laser scanning microscopy using labelled Laponite XLS in the composite hydrogels show two co-continuous areas: red light area and dark area. The tracking of fluorescent microspheres motions in the composite formulations revealed that the red-light area was a dense structure, whereas the dark area was rather loose without aggregated Laponite. This novel special double-network structure facilitates the composite hydrogel to be an adapted biomaterial for specific tissue engineering. Unfortunately, cytotoxicity's assays suggested that XLS Laponites are cytotoxic at low concentration. This study validates that the hybrid interpenetrated network IPN hydrogel has a high modulus that has adapted for tissue engineering, but the cell's internalization of Laponites has to be controlled.

Laponite nanoparticle-associated silated hydroxypropylmethyl cellulose as an injectable reinforced interpenetrating network hydrogel for cartilage tissue engineering.

Articular cartilage is a connective tissue which does not spontaneously heal. To address this issue, biomaterial-assisted cell therapy has been researched with promising advances. The lack of strong mechanical properties is still a concern despite significant progress in three-dimensional scaffolds. This article's objective was to develop a composite hydrogel using a small amount of nano-reinforcement clay known as laponites. These laponites were capable of self-setting within the gel structure of the silated hydroxypropylmethyl cellulose (Si-HPMC) hydrogel. Laponites (XLG) were mixed with Si-HPMC to prepare composite hydrogels leading to the development of a hybrid interpenetrating network. This interpenetrating network increases the mechanical properties of the hydrogel. The in vitro investigations showed no side effects from the XLG regarding cytocompatibility or oxygen diffusion within the composite after cross-linking. The ability of the hybrid scaffold containing the composite hydrogel and chondrogenic cells to form a cartilaginous tissue in vivo was investigated during a 6-week implantation in subcutaneous pockets of nude mice. Histological analysis of the composite constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and collagens. Overall, this new hybrid construct demonstrates an interpenetrating network which enhances the hydrogel mechanical properties without interfering with its cytocompatibility, oxygen diffusion, or the ability of chondrogenic cells to self-organize in the cluster and produce extracellular matrix components. This composite hydrogel may be of relevance for the treatment of cartilage defects in a large animal model of articular cartilage defects. STATEMENT OF SIGNIFICANCE: Articular cartilage is a tissue that fails to heal spontaneously. To address this clinically relevant issue, biomaterial-assisted cell therapy is considered promising but often lacks adequate mechanical properties. Our objective was to develop a composite hydrogel using a small amount of nano reinforcement (laponite) capable of gelling within polysaccharide based self-crosslinking hydrogel. This new hybrid construct demonstrates an interpenetrating network (IPN) which enhances the hydrogel mechanical properties without interfering with its cytocompatibility, O2 diffusion and the ability of chondrogenic cells to self-organize in cluster and produce extracellular matrix components. This composite hydrogel may be of relevance for the treatment of cartilage defects and will now be considered in a large animal model of articular cartilage defects.

Effects of citrate and NaCl on size, morphology, crystallinity and microstructure of calcium phosphates obtained from aqueous solutions at acidic or near-neutral pH.

Precipitation of calcium phosphates occurs in dairy products and depending on pH and ionic environment, several salts with different crystallinity can form. The present study aimed to investigate the effects of NaCl and citrate on the characteristics of precipitates obtained from model solutions of calcium phosphate at pH 6·70 maintained constant or left to drift. The ion speciation calculations showed that all the starting solutions were supersaturated with respect to dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP) in the order HAP>OCP>DCPD. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses of the precipitates showed that DCPD was formed at drifting pH (acidic final pH) whereas poor crystallised calcium deficient apatite was mainly formed at constant pH (6·70). Laser light scattering measurements and electron microscopy observations showed that citrate had a pronounced inhibitory effect on the crystallisation of calcium phosphates both at drifting and constant pH. This resulted in the decrease of the particle sizes and the modification of the morphology and the microstructure of the precipitates. The inhibitory effect of citrate mainly acted by the adsorption of the citrate molecules onto the surfaces of newly formed nuclei of calcium phosphate, thereby changing the morphology of the growing particles. These findings are relevant for the understanding of calcium phosphate precipitation from dairy byproducts that contain large amounts of NaCl and citrate.

Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate.

An injectable composite silanized hydroxypropyl methyl cellulose/biphasic calcium phosphate (Si-HPMC/BCP) has been investigated in humans with promising results. The aim of this study was to evaluate his efficacy for treating periodontal defects (canine fenestration and premolar furcation) in dog models. At 3 months, we observed that bone formation around BCP particles in furcation model is more discernible but not statistically significant in defects filled with Si-HPMC/BCP compared to healing in control. We suggest that BCP particles sustain the bone healing process by osteoconduction, while the Si-HPMC hydrogel enhances intergranular cohesion and acts as an exclusion barrier. Furthermore, bone ingrowth is not so distinctive in superficial defects where the biomaterial appears unstable. These results with Si-HPMC/BCP are encouraging. In addition, this biomaterial is easy to use and simplifies the process of filling periodontal lesions. However, more researches are needed to improve the viscosity and hardness to adjust the material to the specificities of periodontal defects.

Carotid and femoral atherosclerotic plaques show different morphology.

OBJECTIVE: Results of endovascular repair vary according to the arterial bed. We hypothesized that these differences may be related to the plaque features. To explore this hypothesis, we designed a prospective study that compared carotid and femoral atheroma. METHODS AND RESULTS: Patients that underwent femoral or carotid endarterectomy were included in our study. Demographic data and blood sampling were obtained prior to surgery. Plaques were evaluated for AHA grading, calcification and lipid content. Eighty-eight plaques were harvested during this study (45 carotid specimens and 43 femoral specimens). No differences were noted between carotid and femoral groups regarding demographic and biological data. Histological data more frequently showed fibrous cap atheroma in carotid arteries (75%) and fibrocalcific plaques in femoral arteries (93%), p<0.001. Morphological analyses showed a high prevalence of osteoid metaplasia in femoral arteries (63%) compared to carotid arteries (20%, p<0.001). Biochemical analyses were consistent with histological data, showing higher calcium and lesser cholesterol concentrations in femoral than in carotid plaques (p<0.01). CONCLUSIONS: Femoral and carotid plaques showed different morphology in comparable groups of patients.

Na-doped ß-tricalcium phosphate: physico-chemical and in vitro biological properties.

Synthetic calcium phosphate ceramics as ß-tricalcium phosphate (Ca(3)(PO(4))(2); ß-TCP) are currently successfully used in human bone surgery. The aim of this work was to evaluate the influence of the presence of sodium ion in ß-TCP on its mechanical and biological properties. Five Na-doped-ß-TCP [Ca(10.5-x/2)Na(x)(PO(4))(7), 0 ≤ x ≤ 1] microporous pellets were prepared via solid phase synthesis, and their physico-chemical data (lattice compacity, density, porosity, compressive strength, infrared spectra) denote an increase of the mechanical properties and a decrease of the solubility when the sodium content is raised. On the other hand, the in vitro study of MC3T3-E1 cell activity (morphology, MTS assay and ALP activity) shows that the incorporation of sodium does not modify the bioactivity of the ß-TCP. These results strongly suggest that Na-doped-ß-TCP appear to be good candidates for their use as bone substitutes.

Investigation of alendronate-doped apatitic cements as a potential technology for the prevention of osteoporotic hip fractures: critical influence of the drug introduction mode on the in vitro cement properties.

Combination of a bisphosphonate (BP) anti-osteoporotic drug, alendronate, with an apatitic calcium phosphate cement does not significantly affect the main properties of the biomaterial, in terms of injectability and setting time, provided that the BP is introduced chemisorbed onto calcium-deficient apatite, one of the components of the cement. In contrast to other modes of introducing the BP into the cement formulation, this mode allows to minimize alendronate release in the cement paste, thus limiting the setting retardant effect of the BP. An original approach based on high frequency impedance measurements is found to be a convenient method for in situ monitoring of the cement setting reaction. The release profile of the drug from a cement block under continuous flow conditions can be well described using a coupled chemistry/transport model, under simulated in vivo conditions. The results show that the released alendronate concentration is expected to be much lower than the cytotoxic concentration.

Calcium-deficient apatite synthesized by ammonia hydrolysis of dicalcium phosphate dihydrate: influence of temperature, time, and pressure.

In this work, calcium-deficient apatites (CDA) were synthesized by ammonia hydrolysis reaction of dicalcium phosphate dihydrate (DCPD; CaHPO4 x 2 H2O) to obtain biphasic calcium phosphates (BCP) without any extraionic substitution. The influence of three parameters was studied: temperature of the reaction (70 and 100 degrees C), time of the reaction (4 and 18 h), and the pressure (open and closed system). Experiments were made according to a factorial design method (FDM) allowing optimization of the number of samples as well as statistical analysis of results. Moreover, the influence of temperature (until 200 degrees C) was investigated. The crystal size of CDA was determined according to the Scherrer's formula and from Rietveld refinements taking the CDA anisotropy into account. The last method seems to be a reliable method to determine crystallite sizes of CDA, since crystallite sizes of CDA along <00l> and directions were accessible. The results describe the hydroxyapatite % (HA%) in BCP by a first-order polynomial equation in the experimental area studied and the HA content was found to increase by raising time and temperature of the reaction. Moreover, the type of reaction system (open/closed vessel) appeared to have little influence on HA%.

Bone repair using a new injectable self-crosslinkable bone substitute.

A new injectable and self-crosslinkable bone substitute (IBS2) was developed for filling bone defects. The IBS2 consisted of a chemically modified polymer solution mixed with biphasic calcium phosphate (BCP) ceramic particles. The polymer hydroxypropylmethyl cellulose was functionalized with silanol groups (Si-HPMC) and formed a viscous solution (3 wt %) in alkaline medium. With a decrease in pH, self-hardening occurred due to the formation of intermolecular -Si-O- bonds. During setting, BCP particles, 40 to 80 microm in diameter, were added to the polymer solution at a weight ratio of 50/50. The resulting injectable material was bilaterally implanted into critically sized bone defects at the distal femoral epiphyses of nine New Zealand White rabbits. The IBS2 filled the bone defects entirely and remained in place. After 8 weeks, bone had grown centripetally and progressed towards the center of the defects. Newly formed bone, ceramic, and nonmineralized tissue ratios were 24.6% +/- 5.6%, 21.6% +/- 5.8%, and 53.7% +/- 0.1%, respectively. Mineralized and mature bone was observed between and in contact with the BCP particles. The bone/ceramic apposition was 73.4% +/- 10.6%. The yield strength for the IBS2-filled defects was 16.4 +/- 7.2 MPa, significantly higher than for the host trabecular bone tissue (2.7 +/- 0.4 MPa). This study showed that BCP particles supported the bone healing process by osteoconduction while the Si-HPMC hydrogel created intergranular space for bone ingrowth. This new injectable and self-crosslinkable bone substitute could be used conveniently in orthopedic surgery for filling critical-size bone defects.

Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering.

Calcium phosphate ceramics are currently used as bone graft substitutes in various types of clinical applications. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties. By combining these two biomaterials, new composite scaffolds were prepared. In this study, we attempt to analyse whether thrombin concentration in the fibrin glue could influence the properties of the composite. The association between fibrin glue and calcium phosphate ceramic granules was characterized at the ultra structural level. Micro and macroporous biphasic calcium phosphate ceramic granules with a diameter of 1-2mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) were associated to fibrin glue. The composites were observed by scanning and transmission electron microscopy and microcomputed tomography. Fibre thickness, porosity and homogeneity of the fibrin clot were modified by increased the thrombin concentration. Mixing fibrin glue with calcium phosphate granules (1:2) did not modify the microstructure of the fibrin clot in the composite. Nevertheless, thrombin interacted with the bioceramic by inducing the nucleation of crystalline precipitate at the ceramic/fibrin glue interface. Combining fibrin sealant and calcium phosphate ceramics could lead to new scaffolds for bone tissue engineering with the synergy of the properties of the two biomaterials.