José D. Santos

Structural analysis of Si-substituted hydroxyapatite: zeta potential and X-ray photoelectron spectroscopy

The aim of this study was to determine the effect of the incorporation of silicon on the surface charge of hydroxyapatite (HA) and to assess surface structural changes of HA and Si–HA induced by dissolution in both static and dynamic systems. X-ray photoelectron spectroscopy (XPS) analysis showed that SiO44− groups were substituted for PO43− groups in the silicon-hydroxyapatite (Si–HA) lattice according to a previously proposed substitution mechanism without the formation of other crystalline phases, such as tricalcium phosphate or calcium oxide. The substituted silicon induced a decrease in the net surface charge and the isoelectric point of HA as determined by zeta potential (ZP) measurements. At physiological pH=7.4 the surface charge of Si–HA was significantly lowered compared to unmodified HA, i.e. −50±5 to −71±5 eV, caused by the presence of silicate groups in the HA lattice, which may account for a faster in vitro apatite formation using SBF testing. XPS results indicated that silicon seems to be preferentially leached out from Si–HA surface compared to other ionic species after dissolution studies in tris-buffer using a dynamic system.

Preparation and characterization of fluoride-substituted apatites.

Apatites were prepared with three different fluoride concentrations: 0.0 mM (pure hydroxyapatite) 2.5 mM and 5 mM. Reactions were performed in aqueous medium using a reaction between diammonium orthophosphate and calcium nitrate 4-hydrate and ammonium fluoride at temperatures of 3°, 25°, 60° and 90°C. The effects of reaction temperature and fluoride concentration on the crystal morphology, phase purity and crystallinity of the precipitates were observed, using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and ion chromatography. Transmission electron micrographs revealed that the crystallites precipitated at 3°C were spheroidal, but became increasingly acicular with increasing precipitation temperature. X-ray diffraction results indicated that all the materials produced were phase pure and that the crystallinity of apatites prepared at higher precipitation temperatures was higher than those prepared at lower precipitation temperatures. A significant difference in the a-axis dimension of fluoride-substituted apatites was observed, as compared to hydroxyapatite. FTIR spectroscopy revealed a hydroxyl band at 3568 cm-1, along with a broad peak of adsorbed water in the region of 3568 cm-1 to 2670 cm-1 in the hydroxyapatite and fluoride-substituted apatite powders. Hence by careful selection of the precipitation conditions and fluoride contents, the composition and morphology of fluoride-substituted apatite may be controlled and this has interesting implications for the development of these materials for biomedical implantation.

Microstructural characterization of glass-reinforced hydroxyapatite composites

The influence of phosphate-based glasses and a bioactive silica glass on the sintering mechanism of hydroxyapatite was studied over a wide range of temperatures. The composites were microstructurally characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Small additions of phosphate-based glasses proved to be beneficial to the sintering process and fully dense materials were obtained. A significant improvement in mechanical properties was achieved. beta-TCP and alpha-TCP were found in the microstructure depending on the sintering temperature. Additions of bioactive glass led to the development of calcium phosphate silicate.

Hydrophobicity, surface tension, and zeta potential measurements of glass-reinforced hydroxyapatite composites.

Wettability and zeta potential studies were performed to characterize the hydrophobicity, surface tension, and surface charge of P2O5-glass-reinforced hydroxyapatite composites. Quantitative phase analysis was performed by the Rietveld method using GSAS software applied to X-ray diffractograms. Surface charge was assessed by zeta potential measurements. Protein adsorption studies were performed using vitronectin. Contact angles and surface tensions variation with time were determined by the sessile and pendent drop techniques, respectively, using ADSA-P software. The highest (-18.1 mV) and lowest (-28.7 mV) values of zeta potential were found for hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), respectively, with composite materials presenting values in between. All studied bioceramic materials showed similar solid surface tension. For HA and beta-TCP, solid surface tensions of 46.7 and 45.3 mJ/m2, respectively, were obtained, while composites presented intermediate surface tension values. The dispersive component of surface tension was the predominant one for all materials studied. Adhesion work values between the vitronectin solution and HA and beta-TCP were found to be 79.8 and 88.0 mJ/m2, respectively, while the 4.0 wt % glass composites showed slightly lower values than the 2.5 wt % ones. The presence of beta-TCP influenced surface charge, hydrophobicity, and protein adsorption of the glass-reinforced HA composites, and therefore indirectly affected cell-biomaterial interactions.

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.

Adsorption and release studies of sodium ampicillin from hydroxyapatite and glass-reinforced hydroxyapatite composites

As a potential therapy for periodontitis, sodium ampicillin, a broad spectrum antibiotic, was adsorbed onto hydroxyapatite (HA) and glass-reinforced hydroxyaptite (GR-HA) composites, and was subsequently released in vitro. The sodium ampicillin, was adsorbed more on HA compared to the GR-HA composites. X-ray diffraction (XRD) and Rietveld analysis were used to identify and quantify the levels of HA and beta-tricalcium phosphate (beta-TCP) in the microstructure of the GR-HA composites. Lattice parameters changes were observed for the beta-TCP phase dependant on the amount of glass added. The release kinetics were shown to be divided into three stages, the first of which where a large amount of sodium ampicillin is released, followed by a slower release rate and then a final stage where the release amount approaches zero, until no more sodium ampicillin was present. X-ray photoeletron spectroscopy (XPS) studies were carried out in order to ensure that the entire antibiotic adsorbed onto the materials had been released. These kinetics studies have indicated the possibility of using these materials as possible carriers for drug delivery.

Reinforcement of hydroxyapatite by adding P2O5-CaO glasses with Na2O, K2O and MgO

Commercial hydroxyapatite was reinforced by adding small amounts (2 and 4 wt%) of P2O5-based glasses during its sintering process. The composites prepared had a chemical composition closely related to the mineral part of bone tissues in terms of trace elements usually detected, such as Na, K and Mg. X-ray diffraction analysis (XRD) showed that the glass reinforced-HA composites were composed of a HA matrix and variable amounts of tricalcium phosphate phase, depending on sintering temperature and glass composition. These composites were shown to have much higher biaxial bending strength than sintered HA, 107 MPa for Ha/2% of 35P2O5-35CaO-10Na2O-10K2O-10MgO glass composite and 28 MPa for sintered HA. The presence of β-tricalcium phosphate in the microstructure of the composites is an important factor in the reinforcement process.

Physical, chemical and in vitro biological profile of chitosan hybrid membrane as a function of organosiloxane concentration

We attempted to prepare chitosan-silicate hybrid for use in a medical application and evaluated the physico-chemical properties and osteocompatibility of the hybrids as a function of gamma-glycidoxypropyltrimethoxysilane (GPTMS) concentration. Chitosan-silicate hybrids were synthesized using GPTMS as the reagent for cross-linking of the chitosan chains. Fourier transform infrared spectroscopy, (29)Si CP-MAS NMR spectroscopy and the ninhydrin assay were used to analyze the structures of the hybrids, and stress-strain curves were recorded to estimate their Youngs modulus. The swelling ability, contact angle and cytocompatibility of the hybrids were investigated as a function of the GPTMS concentration. A certain fraction of GPTMS in each hybrid was linked at the epoxy group to the amino group of chitosan, which was associated with the change in the methoxysilane group of GPTMS due to hybridization. The cross-linking density was around 80% regardless of the volume of GPTMS. As the content of GPTMS increased, the water uptake decreased and the hydrophilicity of the hybrids increased except when the content exceeded amolar ratio of 1.5, when it caused a decrease. The values of the mechanical parameters assessed indicated that significant stiffening of the hybrids was obtained by the addition of GPTMS. The adhesion and proliferation of the MG63 osteoblast cells cultured on the chitosan-GPTMS hybrid surface were improved compared to those on the chitosan membrane, regardless of the GPTMS concentration. Moreover, human bone marrow osteoblast cells proliferated on the chitosan-GPTMS hybrid surface and formed a fibrillar extracellular matrix with numerous calcium phosphate globular structures, both in the presence and in the absence of dexamethasone. Therefore, the chitosan-GPTMS hybrids are promising candidates for basic materials that can promote bone regeneration because of their controllable composition (chitosan/GPTMS ratio).

Crystallization and microstructure analysis of calcium phosphate-based glass ceramics for biomedical applications

Abstract Calcium phosphate glasses and glass ceramics (CaO/P 2 O 5 =1.25 in molar ratio) modified by small amounts of additives such as Na 2 O, MgO, TiO 2 and ZrO 2 were prepared after appropriate heat treatment for nucleation and crystallization. Based upon differential thermal analysis (DTA) results, a two-step heat treatment was used for crystal nucleation and growth. X-ray diffraction (XRD) analysis demonstrated that bioresorbable β-Ca 2 P 2 O 7 (β-DCP), β-Na 2 CaP 2 O 7 , Na 2 Mg(PO 3 ) 4 phases were formed in the glass matrix depending upon the relative contents of the additives. By adding higher contents of Na 2 O and TiO 2 and using CaO/P 2 O 5 =1.5–2.0, crystallization of β-DCP and Ca 3 (PO 4 ) 2 (β-TCP), and the formation of a dense structure in the glass ceramics were obtained. The precipitation of these crystals could be well distinguished at a magnification of 4000× and phases were dispersed in areas of micron size. A porous structure may be easily formed after the soluble phases are dissolved in physiological media. These glass ceramics with high CaO/P 2 O 5 ratio, modified by the above mentioned additives are expected to find use as implants for bone replacement/regeneration and drug delivery carriers synergistically, because the soluble phases may act as drug delivery carriers and the porous structure will allow for bone ingrowth.