Glass-reinforced Hydroxyapatite Composites Research Articles

Novel Bioceramic Scaffolds for Regenerative Medicine

Calcium phosphate ceramics have been used as synthetic grafts for bone repair. This family of alloplastic grafts is an alternative to allografts (from other individuals from the same specie), autografts (from the same individual) or xenografts (from individuals from other species). Sintered bovine bone is basically composed mainly by hydroxyapatite (HA), Ca10(PO4)6(OH)2 but chemical analyses indicate the presence of Mg. Chemical and heat treatments are generally required to eliminate biological hazard. However, the more crystalline hydroxyapatite, the less resorbable the product is. An approach to have a highly crystalline and still resorbable material is to use additions of alpha or beta tricalcium phosphate, Ca3(PO4)2 , (TCP). The addition of fractions of some bioactive glasses to hydroxyapatite has shown to be effective in promoting its decomposition to tricalcium phosphate. In addition, glass reinforced hydroxyapatite composite are materials with higher compressive strength due to liquid phase sintering. In this study, novel scaffolds based on hydroxyapatite and tricalcium phosphate are presented.

The benefit of a human bone marrow stem cells concentrate in addition to an inorganic scaffold for bone regeneration: an in vitro study.

Background. This work compares the osteoblastic behaviour of a bone marrow (BM) aspirate and a prepared BM concentrate of nucleated cells associated with a glass reinforced hydroxyapatite composite (GRHC) in a microporous pellet formulation. Methods. BM aspirate (30 mL) was collected during 3 orthopedic surgical procedures, and a concentration system was used to achieve 3 rapid preparations of a concentrate of nucleated cells (3 mL) from the BM aspirates. The BM aspirates (53% cell viability; 2.7 × 106 nucleated cell/mL) and the BM concentrates (76% cell viability; 2 × 107 nucleated cell/mL) were cultured over glass reinforced hydroxyapatite pellets, at the same volume/mass ratio, for 30 days. Cultures performed in standard tissue culture plates were used as control. Results. The colonized BM concentrate/material constructs exhibited a representative osteoblastic proliferation/differentiation pathway, evidenced by a high alkaline phosphatase (ALP) activity, expression of collagen type 1, ALP, BMP-2, M-CSF, RANKL, and OPG, and formation of a calcium phosphate mineralized matrix. A clear improved behaviour was noticed compared to the BM aspirate/material constructs. Conclusions. The results suggest the benefit of using an autologous BM concentrate/material construct in the clinical setting, in bone regeneration applications.

Morphology effect of bioglass-reinforced hydroxyapatite (Bonelike(®) ) on osteoregeneration.

In the last decades, the well-known disadvantages of autografts and allografts have driven to the development of synthetic bone grafts for bone regeneration. Bonelike(®) , a glass-reinforced hydroxyapatite (HA) composite was developed and registered for bone grafting. This biomaterial is composed by a modified HA matrix, with α- and β-tricalcium phosphate secondary phases. Aiming to improve the biological characteristics of Bonelike(®) , new spherical pelleted granules, of different shape and size, were developed with controlled micro and macrostructure. In the present study, it was compared the physicochemical properties and in vivo performance of different Bonelike(®) granule presentations-Bonelike(®) polygonal (500-1000 µm size) and Bonelike spherical (250-500 µm; 500-1000 µm size). For the in vivo study, Bonelike(®) was implanted on sheep femurs, with various implantation times (30 days, 60 days, 120 days, and 180 days). X-ray diffraction analysis revealed that the phase composition of different granules presentations was similar. Bonelike(®) spherical 500-1000 µm was the most porous material (global porosity and intraporosity) and Bonelike(®) polygonal 500-1000 µm the less porous. Considering the in vivo study, both polygonal and spherical granules presented osteoconductive proprieties. The spherical granules showed several advantages, including easier medical application through syringe and improved osteointegration, osteoconduction, and degradation, by the presence of larger pores, controlled micro- and macrosctructure and suitable particle format that adapts to bone growth. Bonelike(®) spherical 500-1000 µm showed improved new bone invasion throughout the material's structure and Bonelike(®) spherical 250-500 µm appeared to induce faster bone regeneration, presenting less unfilled areas and less lacunae in the histological analysis.

Growth and phenotypic expression of human endothelial cells cultured on a glass-reinforced hydroxyapatite

Glass-reinforced hydroxyapatite composites (GR-HA) are bone regenerative materials that are characterized by their increased mechanical properties, when compared to synthetic hydroxyapatite. Bonelike is a GR-HA that is a result of the addition of a CaO-P(2)O(5) based glass to a HA matrix. This biomaterial has been successfully applied in clinical bone regenerative applications. This work aims to evaluate the ability of Bonelike to support the adhesion, proliferation and phenotypic expression of human endothelial cells, aiming to establish new bone tissue engineering pre-endothelialization strategies. Bonelike discs, regardless of being submitted to a pre-immersion treatment with culture medium, were seeded with first passage human umbilical vein endothelial cells, and characterized regarding proliferation and differentiation events. Pre-immersed Bonelike allowed the adhesion, proliferation and phenotype expression of endothelial cells. Seeded materials presented positive immunofluorescent staining for PECAM-1 and a tendency for the formation of cord-like arrangements under angiogenesis-stimulating conditions, although, compared to standard culture plates, a slight decreased cell growth was observed. In this way, Bonelike may be a suitable candidate for pre-endothelialization approaches in bone tissue engineering applications.

Clinical applications of Titanium dental implants coated with glass-reinforced Hydroxyapatite composite (Bonelike®)

Commercially pure titanium dental implants were coated with glass-reinforced hydroxyapatite composites (Bonelike?) using the plasma spraying technique. A total of 27 Bonelike? coated 3.75 mm diameter and 10 mm length implants were placed in the maxilla (18) and mandible (9) of seven patients. Pre and post-operative radiological examination was performed by orthopantomogram according to the standard follow-up protocols. After a 3-month healing period, one patient's implant from the mandible was surgically removed due to bad positioning and was then assessed by light and scanning electron microscopy for histological analysis. The reported Bonelike? coated dental implants proved to be highly bioactive with extensive new bone formation and attachment.

In Vitro Assessment of New Niobium Phosphate Glasses and Glass Ceramics

Niobo-phosphate glasses were produced in order to assess the citotoxicity of samples with different compositions. Different P2O5/Ca ratios were studied in an attempt to correlate the biological behavior of new niobo-phosphate bioactive glasses with conventional bioactive calcium phosphate glasses. The most biocompatible glass composition was chosen to produce glassreinforced hydroxyapatite composites.

Production of Porous Biomaterials Based on Glass-Reinforced Hydroxyapatite Composites

Production of Porous Biomaterials Based on Glass-Reinforced Hydroxyapatite Composites

Sintering effects on mechanical properties of glass-reinforced hydroxyapatite composites

The improvement of mechanical properties of hydroxyapatite (HA) with reinforcement of two (45.5 wt.% P 2O 5, 54.5 wt.% CaO) and three-oxide (45 wt.% P 2O 5, 28 wt.% CaO, 27 wt.% Na 2O) glasses with different sintering temperatures is studied. Although HA shows great biocompatibility with the human body, its applications are limited to non load-bearing areas and coatings due to its low mechanical properties. These mechanical properties can be improved substantially with addition of glass ceramics by sintering. In this study, naturally produced HA from human teeth is sintered with varying addition of two and three-oxide glass ceramics. The microhardness measurements, the density and the compression tests are performed in order to find the optimum sintering temperature. Finally average hardness value of 285±19 HV, average density of 2.79±0.05 g cm −3 and compressive strength value of σ avr=67±17 MPa have been achieved by sintering at 1200 °C with the addition of 10 wt.% two oxide glass into the HA. Results are implying that 10 wt.% of glass addition for HA composites is the maximum limit for such composites. Observations are supporting the idea that sodium free glasses could be better conciliated with the HA structure at high temperatures.

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 β-tricalcium phosphate ( β-TCP) in the microstructure of the GR-HA composites. Lattice parameters changes were observed for the β-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.

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

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

Structural insights of glass-reinforced hydroxyapatite composites by Rietveld refinement

Phase transformations and interstitial and/or substitution of trace elements during the liquid-phase sintering process of P 2O 5–CaO–MgO glass-reinforced hydroxyapatite (GR-HA) composites were examined by X-ray diffraction and Rietveld analyses. Using the Rietveld method for structure refinement, changes in the lattice parameters of the two main phases of the composites, hydroxyapatite (HA) and β-tricalcium phosphate ( β-TCP), as well as changes in several bond lengths and in the occupancy of the hydroxyl oxygen site in the HA phase structure were assessed. The glasses gave rise to formation of between approximately 45 and 50% of β-TCP, with evidence for the Mg 2+ enhancing the formation of β-TCP. Between 1300 and 1350°C, the β-TCP inverts to α-TCP, without further decomposition of the residual HA. The glasses showed evidence for stabilisation of the hydroxyl group located in the hydroxyl channels. This is supported by measurements of the hydroxyl channel radius ( R c), the Ca 2–OH bond length and the hydroxyl oxygen occupancy ( O occ). Results showed that the Mg 2+ containing glasses induced the β-TCP phase formation in the structure of GR-HA composites and retarded the β-TCP into α-TCP transformation at higher temperatures. The chemical composition of the P 2O 5 glasses also induces modifications in the lattice parameters of the crystallographic phases present in the microstructure of the composites. This suggests some substitution of Mg 2+-for-Ca 2+ in the β-TCP structure during the liquid-phase sintering process.

Direct and indirect effects of P 2O 5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells

Human osteoblast-like cells were plated on hydroxyapatite and P 2O 5-glass reinforced hydroxyapatite composite discs. They were also cultured in the presence of media obtained by incubating the discs in the absence of cells. The effects of these direct and indirect interactions were examined by measuring cell proliferation and the expression of certain key extracellular matrix antigens. One composite was found to initially delay cell growth, while the extract of a different composite appeared to down-regulate DNA synthesis. Flow cytometry analysis showed that growth directly on the discs had little effect on collagen type I, but reduced fibronectin and osteocalcin levels. The extracts of the materials generally had less effect, although one extract obtained from the glass-reinforced hydroxyapatite significantly down-regulated fibronectin. These in vitro studies thus suggest that there were only few differences overall in the growth of the cells directly on the glass-reinforced compared with the hydroxyapatite discs and also only relatively small effects of the extracts on the cells. However, the flow cytometry results suggest that both the materials and the extracts may have a potentially important influence on connective tissue production, and that these effects are both material- and antigen-specific.

Microstructural dependence of Young's and shear moduli of P 2O 5 glass reinforced hydroxyapatite for biomedical applications

P 2O 5 glass reinforced hydroxyapatite composite materials were prepared through a liquid-phase sintering process. Secondary phases, β- and α-tricalcium phosphates ( β-TCP and α-TCP), were formed in the microstructure of the composites, due to the reaction between the liquid glassy phase and the hydroxyapatite matrix. The dynamic Young's 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 β-TCP and α-TCP phases and with the porosity percentage present in the microstructure. Glass reinforced hydroxyapatite composites showed lower Young's and shear moduli than unmodified hydroxyapatite, mainly due to the presence of β-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.

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.

Glass-reinforced hydroxyapatite composites: fracture toughness and hardness dependence on microstructural characteristics.

Fracture toughness and hardness properties of CaO-P2O5 glass-reinforced hydroxyapatite composites have been assessed using indentation techniques and results calculated according to Laugier and Evans' equations. Both properties showed to be dependent upon several microstructural characteristics, namely residual porosity and the percentage of secondary beta and alpha tricalcium phosphate phases in the structure of the composites. Composites presented a Palmqvist-type indentation crack system, which is the specific crack system addressed by Laugier's approach. Fracture toughness determinations according to Evan's equation, which is a universal one and adapted to both median and Palmqvist crack systems, did not correlate well with Laugier determinations.

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.

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.

In vitro calcium phosphate formation on SiO2-Na2O-CaO-P2O5 glass reinforced hydroxyapatite composite: a study by XPS analysis

In-vitro apatite layer formation on the surface of a newly developed glass reinforced hydroxyapatite composite was characterized using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) equipped with energy dispersive X-ray analysis (EDXA). After 1 week soaking in an acellular simulated body fluid the composite surface was entirely covered by a Ca, P-layer, suggesting a bioactive behaviour. XPS binding energy results revealed that this surface layer was a carbonated and non-stoichiometric apatite with Ca/P ratio of 1.3. This apatite layer was composed of very fine needle-like crystallites. Comparative studies on a commercially available hydroxyapatite showed that a similar apatite layer was also formed on its surface.

Surface modifications of glass-reinforced hydroxyapatite composites

Surface modifications of glass-reinforced hydroxyapatite composites immersed in a simulated physiological solution were studied using X-ray photoelectron spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. In the first stages of the apatite formation process, calcium and silicon ions were leached out from the surface of the composites. After 12 days of immersion, apatite crystals were detected on the surface indicating bioactive behaviour. Sodium ions attributed to sodium sulphate were also found in the apatite layer. Bound water on the surface also increased with immersion time.

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. β-TCP and α-TCP were found in the microstructure depending on the sintering temperature. Additions of bioactive glass led to the development of calcium phosphate silicate.