Antonio J. Salinas

Bioactive sol-gel glasses with and without a hydroxycarbonate apatite layer as substrates for osteoblast cell adhesion and proliferation.

The biocompatibility of three sol-gel bioactive glasses with SiO(2) as the main constituent (75, 72.5 and 70 mol%), identical CaO content (25mol%), and without or with P(2)O(5) as third constituent (0, 2.5 and 5 mol%), have been analyzed (S75, S72.5P2.5, and S70P5 glasses). These studies were performed on both untreated glasses and on glasses coated with a hydroxycarbonate apatite (HCA) layer formed in vitro by soaking 7d in an acellular simulated body fluid. Cell attachment, spreading and proliferation were studied using neonatal rat calvaria osteoblasts. Cells attach to the three untreated glasses but show a higher efficiency on that with the higher phosphate content (S70P5). The formation of the HCA layer significantly enhances this process (1.7-fold). In all cases, attachment is followed by cell spreading on the surface of the materials, adopting the cells a flattened morphology and showing diverse anchoring cell projections. Mitotic activity has been detected on osteoblasts growing on the sol-gel glasses, being this process 2-4-fold higher when the apatite-like layer is already formed. Taking into account the results herein presented, these bioactive glasses can be considered biocompatible. In addition, their biocompatibility is greatly enhanced after induction of the formation of an HCA layer.

In vitro calcium phosphate layer formation on sol‐gel glasses of the CaO‐SiO2 system

A glass with a composition of SiO(2) 80% and CaO 20% (in mol %) was prepared by the sol-gel method, and its in vitro bioactivity was studied by soaking it in simulated body fluid (SBF) at 37 degrees C. The formation of a calcium phosphate layer on the glass was analyzed by determining the ionic concentrations in solution and by studying the surface of the glass with X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM), and electron diffraction (ED) after it had been in SBF for varying periods of time. The composition of this bioactive glass, formed of only two components, allowed us to monitor the formation process of the hydroxycarbonate apatite (HCA) layer. The bioactive behavior of this glass indicates that the presence of phosphorous in the glass composition is not an essential requirement for the development of a HCA layer. In this case, the layer is formed because of the phosphorous present in the in vitro assay solution.

Effect of magnesium content on the in vitro bioactivity of CaO-MgO-SiO2-P2O5 sol-gel glasses

Three glasses in the system CaO-MgO-SiO 2 -P 2 O 5 , with high silica content (80 mol% SiO 2 ) and 0, 3 and 7 mol% of MgO, respectively, have been prepared by the sol-gel method. The in vitro bioactivity of pellets, prepared by compacting the glass powders, was assessed by determining the changes in surface morphology and composition after soaking in a simulated body fluid (SBF) for up to 14 days at 37 °C. Formation of a calcium phosphate rich layer on the glasses surface was followed by scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). Three effects of MgO in the glass upon the newly formed layer were detected: (i) it slows down the rate of formation of the layer, (ii) it increases the thickness of the layer and (iii) a whitlockite-like phase appeared together with the apatite-like phase formed on the surface of bioactive materials when exposed to physiological solutions.

A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants

The aim of this study was to propose and validate a new unified method for testing dissolution rates of bioactive glasses and their variants, and the formation of calcium phosphate layer formation on their surface, which is an indicator of bioactivity. At present, comparison in the literature is difficult as many groups use different testing protocols. An ISO standard covers the use of simulated body fluid on standard shape materials but it does not take into account that bioactive glasses can have very different specific surface areas, as for glass powders. Validation of the proposed modified test was through round robin testing and comparison to the ISO standard where appropriate. The proposed test uses fixed mass per solution volume ratio and agitated solution. The round robin study showed differences in hydroxyapatite nucleation on glasses of different composition and between glasses of the same composition but different particle size. The results were reproducible between research facilities. Researchers should use this method when testing new glasses, or their variants, to enable comparison between the literature in the future.

Influence of P2O5 on crystallinity of apatite formed in vitro on surface of bioactive glasses

Two sol-gel glasses with 80 mol % SiO2 were prepared in the system SiO2-CaO-P2O5; the first one had 3 mol% P2O5 in its composition, and the second one was P2O5 free. The in vitro behavior of glasses was studied by soaking them in simulated body fluid for 7 days at 37 degrees C. After the in vitro test, the study by Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, electron diffraction, and transmission electron microscopy showed an apatite-like layer had formed on the surface of both glasses. However, for identical soaking time, the apatite crystals formed on the surface of the glass containing P2O5 in the composition were larger. Therefore, the presence of P2O5 in the sol-gel glass composition promotes the crystal growth of the apatite.

Substitutions of cerium, gallium and zinc in ordered mesoporous bioactive glasses.

Ordered mesoporous glasses based on the 80% SiO(2)-15% CaO-5% P(2)O(5) system including up to 3.5% Ce(2)O(3), 3.5% Ga(2)O(3) or 7.0% ZnO (in mol.%) were synthesized by the evaporation-induced self-assembly process using Pluronic® 123 as a surfactant. An ordered hexagonal mesophase was observed in both the unsubstituted glass (denoted in this paper as B: blank) and glasses containing <0.4% of substituent by X-ray diffraction, transmission electron microscopy and electron diffraction. The increase in the amount of substituent led to a decrease in the mesopore order. B glass exhibited good textural properties: S(BET)=515m(2)g(-1), D(P)=4.7nm and V(P)=0.58cm(3)g(-1). With the inclusion of cerium, gallium and zinc oxides the textural properties decreased, but remained in amounts useful for clinical applications. Zinc-containing samples showed the highest decrement in the textural properties. Substituted glasses exhibited a quick in vitro bioactive response except when the ZnO content was over 0.4%. Taking into account the ordered mesoporosity, the quick in vitro bioactive response and the added values of the substituents, this new family of glasses are promising candidates for applications in bone tissue engineering.

Biomimetic Apatite Deposition on Calcium Silicate Gel Glasses

In order to understand the Biomimetic apatite formation mechanism on gel glasses, a glass (in mol-%) SiO2 80%—CaO 20% (80S20C) was prepared by the sol-gel method and its behaviour in a simulated body fluid (SBF) was studied. To study the role of phosphorous in the in vitro apatite formation, a gel glass (in mol-%) SiO2 80%—CaO 17%—P2O5 3% (80S17C3P) was prepared comparing its behaviour in SBF with that of 80S20C. In both studies, a protocol without renovation of SBF (static) was used. To mimic the conditions in the living organisms, an in vitro protocol with continuous renovation of solution (dynamic) was proposed. To check the feasibility of dynamic protocol, 80S20C and 80S17C3P were studied in dynamic and results compared with obtained in static. Static studies of 80S20C allowed us to verify that phosphorus is not essential for bioactivity because the apatite-like layer was formed from the phosphorous in SBF. However, a 3 mol-% of P2O5 in 80S17C3P gel glass favoured apatite crystallization. In dynamic, complete assays were performed with ionic concentrations and pH in solution almost equal to human plasma. After 7 days in dynamic, apatite crystals and crystalline aggregates were larger than in static. Besides, compositional variations were observed in the newly formed layer as a function of the protocol. In static, the layer formed in both glasses contained calcium and phosphorous, (Ca/P molar ratio = 1.6) and silicon. In dynamic, the layer did not contain silicon and the Ca/P molar ratio was 1.2. Differences in composition and pH of assay solution, 8 in static and 7.3 in dynamic could explain these variations. In static, an apatite close to stoichiometric could be formed. In dynamic, a mixture of calcium deficient apatite and other calcium phosphates could constitute the layer.

Influence of composition and surface characteristics on the in vitro bioactivity of SiO2−CaO−P2O5−MgO sol-gel glasses

Glasses in the system SiO(2)-CaO-P(2)O(5)-MgO were prepared by the sol-gel method. These glasses featured SiO(2) contents in the range 60-80 mol %, 4 mol % of P(2)O(5), and a CaO/MgO molar ratio of 4. Because of their composition and surface properties, all the glasses showed in vitro bioactivity, as evidenced by the formation of an apatite-like layer on their surface when soaked in an acellular medium with ionic composition similar to human blood plasma. An increase in the CaO content of the glasses also caused an increase in their porosity. Higher porosity facilitated the apatite nucleation on the sample surface during the first days of the in vitro test. On the other hand, those glasses with higher SiO(2) content also showed higher surface area values, as well as higher calcium phosphate layer growth rates. For longer soaking periods, the grown layer was analyzed, revealing a two-phase composition: apatite and whitlockite.

Bioactive ceramics: from bone grafts to tissue engineering

Bioactive ceramics bond directly with living tissues when implanted. For this reason they have been profusely investigated as biomaterials. The first synthetic bioactive materials were specific compositions of glasses and glass ceramics as well as sintered hydroxyapatite. However, all these bioceramics are brittle, and for this reason their main application for years has been as a grafting material for the filling of small bone defects and periodontal anomalies. The efforts to expand the applications of bioactive bioceramics were mainly focused in two areas: (A) the synthesis of organic–inorganic hybrids to apply in tissue engineering and of ceramic coatings on metallic substrates for applications requiring good mechanical behavior, and (B) the synthesis of porous materials with very quick bioactive response that can be upgraded by adding biomolecules or therapeutic inorganic ions to be used in bone tissue engineering. For these developments, the in vitro studies in solutions mimicking blood plasma played a major role. At the present, it is universally considered that both bioactive and biodegradable materials are going to play a central role in the fabrication of porous scaffolds that after being decorated with cells and signals form constructs: basic elements of tissue engineering. This article reviews the pathway followed by the bioactive materials from their original applications in bone grafts to the present day where they are widely investigated as porous scaffolds for bone tissue engineering. After defining the concept of bioactivity, important bioactive materials will be listed in this article. Then, the specific characteristics of bioactive materials when used in bulk or coatings as well as the comparison with biodegradable materials will be presented. Finally, and after describing the in vitro studies for the evaluation of bioactive ceramics, the main characteristics of template glasses, compared with conventional sol–gel glasses, and the advantages of using porous bioactive ceramics to obtain scaffolds for bone tissue engineering will be explained.

The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107-111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using μCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107-111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material.