Maria A. Lopes

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.

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.

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).

Si3N4-bioglass composites stimulate the proliferation of MG63 osteoblast-like cells and support the osteogenic differentiation of human bone marrow cells☆

The invitro osteocompatibility of a n ovel Si 3N4-bioglass composite (70–30% weight proportion) with improved mechanical properties (fracture toughness=4.4 MPa m 1/2 ; bending strength=383747 MPa) is reported. Immersionof the composite samples in culture medium (30 min to 7 days) resulted in rapid protein adsorption to the surface and, also, dissolution of the intergranular phase of bioglass (time-dependent process) with the formation of different size cavities. ‘‘As-received’’ and pre-treated material samples presented a similar behaviour concerning the proliferation of MG63 osteoblast-like cells, evaluated during a 5-day culture period. Seeded materials showed a higher cell growth rate as compared to cultures performed onthe standard plastic culture plates. To assess the osteogenic potential of the composite, ‘‘as-received’’ material samples were seeded with human bone marrow cells and cultured for 35 days in experimental conditions that favour the development of the osteoblastic phenotype. The cell adhesion process was similar to that observed in control cultures. Cells successfully adapted to the irregularities of the surface and were able to grow towards inside the cavities; in addition, osteogenic differentiation occurred with the formation of abundant cell-mediated mineralised deposits. Results suggest that this Si3N4-bioglass composite seems to be a promising candidate for high-stress medical applications.

Push-out testing and histological evaluation of glass reinforced hydroxyapatite composites implanted in the tibia of rabbits

In vitro and in vivo bioactivity studies were performed to assess the biocompatibility of CaO-P2O5 glass-reinforced hydroxyapatite (GR-HA) composites. The ability to form an apatite layer by soaking in simulated body fluid (SBF) was examined and surfaces were characterized using FTIR reflection and thin-film X-ray diffraction analyses. Qualitative histology, histomorphometric measurements, and push-out testing were performed in a rabbit model for characterizing bone/implant bonding. Under the in vitro conditions using SBF, an apatite layer could not be formed on GR-HA composites within 8 weeks. Results of push-out testing showed bonding between the composites and bone, ranging from 130-145 N after 2 weeks of implantation. After the longest implantation period, 16 weeks, the GR-HA composite prepared with the higher content of CaO-P2O5 glass showed the highest bonding force, 606 +/- 45 N, compared to 459 +/- 30 N for sintered HA. Development of immature bone and modifications in the turnover of a more mature bone on the surface of GR-HA composites were similar to those on sintered HA.

Densification route and mechanical properties of Si3N4–bioglass biocomposites

The processing route and the final microstructural and mechanical characteristics of a novel biomaterial composite are described. This new material is composed of 70 wt% Si3N4 ceramic phase and 30 wt% bioglass, the later performing as a liquid sintering aid system and simultaneously providing bioactivity characteristics to the composite. The conditions for fabrication of an almost fully dense material (approximately 98% of relative density) were pursued. Optimised parameters were 1350 degrees C-40 min-30 MPa by hot-pressing technique. The very fast densification rate of the process avoided the crystallisation of the bioglass intergranular phase and therefore its intrinsic properties were maintained. Also, the large amount of glassy phase assured the densification by liquid phase assisted grain rearrangement without Si3N4 phase transformation. The final mechanical properties of the Si3N4 bioglass were as follows: fracture toughness, K(IC) = 4.4 MPa m(1/2); Vickers hardness, Hv = 10.3 GPa; Youngs modulus, E = 197 GPa; bending strength, sigma(g) = 383 MPa; Weibull modulus, m = 8.3. These values provide an attractive set of properties among other bioactive materials, namely by upgrading the main drawback of bioceramcs and bioglasses for high-load medical applications, which is the lack of satisfactory fracture toughness.

Microstructural dependence of Young's and shear moduli of P2O5 glass reinforced hydroxyapatite for biomedical applications

P2O5 glass reinforced hydroxyapatite composite materials were prepared through a liquid-phase sintering process. Secondary phases, beta- and alpha-tricalcium phosphates (beta-TCP and alpha-TCP), were formed in the microstructure of the composites, due to the reaction between the liquid glassy phase and the hydroxyapatite matrix. The dynamic Youngs modulus (E) and shear modulus (G) of these composites were determined using an impulse excitation method. By applying the Duckworth-Knudsen equation, the elastic property results were correlated with the relative proportion of beta-TCP and alpha-TCP phases and with the porosity percentage present in the microstructure. Glass reinforced hydroxyapatite composites showed lower Youngs and shear moduli than unmodified hydroxyapatite, mainly due to the presence of beta-TCP phase. The Duckworth-Knudsen model demonstrated an exponential dependence of E and G modulus with porosity and mathematical equations were derived for composite materials with porosity correction factors (b) of 4.04 and 4.11, respectively, indicating that porosity largely decreased both E and G moduli.

Cytotoxicity evaluation of nanocrystalline diamond coatings by fibroblast cell cultures

The cytotoxicity profile of nanocrystalline diamond (NCD) coatings on a Si(3)N(4) ceramic was investigated. This material is envisaged to have biomedical dental applications such as burrs and surgical instruments. Two fibroblast cell culture systems were used to address the cytotoxicity of NCD-coated samples: L929 cells (a mouse permanent cell line) and human gingival fibroblasts. Cell behavior was evaluated in terms of cell adhesion, cell viability/proliferation (mitochondrial function, MTT assay) and the pattern of cell growth. Fibroblast cell behavior on standard polystyrene culture plates was used as control, as Si(3)N(4) substrates have previously been shown to be biocompatible. NCD coatings provided a suitable surface for cell attachment, spreading and proliferation. Human gingival cells showed a homogeneous cytoplasm spreading, a flattened elongated morphology and a typical parallel alignment on confluent cultures. In comparison, L929 cells denoted a lower cytoplasm expansion, a heterogeneous spreading but a higher proliferation rate. For both cells, after few days, the NCD coating was completely covered with continuous cell layers. As compared to standard polystyrene culture plates, no deleterious or cytotoxic responses were observed with L929 and human fibroblast cell cultures, and in both a slight enhancement in cell proliferation was observed. In addition, the seeded NCD film allowed reproduction of the typical features of the two cell culture systems tested, further suggesting the lack of cytotoxicity of this coating.

Glass-reinforced hydroxyapatite composites: Secondary phase proportions and densification effects on biaxial bending strength

CaO-P(2)O(5) glasses with additions of MgO and CaF(2) were used as a sintering aid of hydroxyapatite, and glass-reinforced hydroxyapatite composites obtained. Glasses promoted significant changes in the microstructure of the composites, namely with the formation of tricalcium phosphate secondary phases, beta and alpha-TCP. Quantitative phase analysis was performed by the Rietveld method using General Structure Analysis Software. Grain size measurements were carried out on SEM photomicrographs, using a planimetric procedure according to ASTM E 112-88. Flexural bending strength was determined from concentric ring-on-ring testing. Flexural bending strength (FBS) of glass-reinforced hydroxyapatite composites was found to be about twice or three times higher than that of unreinforced hydroxyapatite and tended to depend more on porosity and beta and alpha-TCP secondary phases, rather than on grain size. Traces of alpha-tricalcium phosphate significantly enhanced the strength of the composites. Using the rule of mixtures to estimate the zero porosity bending strength, the Duckworth-Knudsen model applied to the composites gave a porosity correction factor, b, with a value of 4.02. Weibull statistics were also used to analyze biaxial strength data and the level of reinforcement obtained by comparing failure probability for the composites and for the unreinforced hydroxyapatite. Lower activation energies for grain growth were observed for the composites compared to unreinforced hydroxyapatite, which should be attributed to the presence of a liquid glassy phase that promotes atomic diffusion during the sintering process.