Key Engineering Materials Vols. 493-494

Abstract: Basic drawbacks of calcium phosphate cements (CPCs) are the brittleness and low strength behavior which prohibit their use in many stress-bearing locations, unsupported defects, or reconstruction of thin bones. Recently, to solve these problems, researchers investigated the incorporation of fibers into CPCs to improve their strength. In the present study, various amounts of a highly bioactive glass fiber were incorporated into calcium phosphate bone cement. The obtained results showed that the compressive strength of the set cements without any fibers optimally increased by further addition of the fiber phase. Also, both the work-of-fracture and elastic modulus of the cement were considerably increased after applying the fibers in the cement composition. Herein, with the aim of using the reinforced-CPC as appropriate bone filler, the prepared sample was evaluated in vitro using simulated body fluid (SBF) and osteoblast cells. The samples showed significant enhancement in bioactivity within few days of immersion in SBF solution. Also, in vitro experiments with osteoblast cells indicated an appropriate penetration of the cells, and also the continuous increase in cell aggregation on the samples during the incubation time demonstrated the ability of the reinforced-CPC to support cell growth. Therefore, we concluded that this filler and strong reinforced-CPC may be beneficial to be used as bone fillers in surgical sites that are not freely accessible by open surgery or when using minimally invasive techniques.

Abstract: Dense and submicron-sized hydroxyapatite powders were synthesized through spray pyrolysis of calcium phosphate solution with polyethylene glycol (PEG). Irregular shaped and fragile hydroxyapatite aggregates, and spherical and hollow hydroxyapatite particles were obtained after spray pyrolysis at 1100 °C when using calcium phosphate solution with and without PEG, respectively. After dewaxing at 650 °C for 3 hours, the hydroxyapatite aggregates were broken into submicron-sized particles whereas micron-sized spherical hollow particles were almost intact. The relative densities of hydroxyapatite disks made by submicron and micron sized hydroxyapatite particles were about 90% and 81%, respectively, after sintering at 1110 °C for 3 hours. The results were explained in terms of the disintegrating function of carbon which was previously formed among the hydroxyapatite aggregates by the decomposition of PEG during spray pyrolysis.

Abstract: Phase-pure α-TCP powder was milled using a high-energy planetary mill to obtain a partially X-ray amorphous material. Calcination at temperatures between 350 and 600 °C was employed to recrystallize the powder. The phase composition as a function of calcination time and temperature was determined in-situ using high-temperature XRD equipment. It was found that the amorphous fraction recrystallized mainly to α-TCP, with only small amounts of β-TCP formed. At low temperatures (≤ 450 °C), a stable composition with approximately 85 wt-% α-TCP was found once 100% crystallinity was reached. The time required to reach full crystallinity depended on the calcination temperature. For temperatures > 450 °C a slow transformation to β-TCP was observed. The transformation rate depended on the calcination temperature and on the milling intensity. A moderately milled powder recrystallized to α-TCP, followed by a slow transformation to β-TCP at 600 °C, whereas an intensely milled powder also recrystallized to α-TCP, followed by a fast transformation to β-TCP at the same temperature.

Abstract: Several substituted â-tricalcium phosphates have been prepared with different cations (monovalent, divalent and trivalent) and at various levels of substitution. Structural investigations have proved that fewer than ~10% wt substitution, the substituted compounds are isostructural to â- TCP, leading to solid solutions. These samples have been characterized by infrared and Raman spectroscopies. The vibrational spectra show mainly the bands related to the vibrations of PO43- tetrahedrons present in the structure. As Raman scattering and infrared absorption are local probes, the bands are sensitive to the local environment of the distinct tetrahedrons, related to the site of substitution and to the nature of the cations.

Abstract: Nanocrystalline carbonated hydroxyapatite was produced through hydraulic conversion of calcium phosphate cement in simulated body fluid (SBF) and then heated in a microwave oven at 1000-1250 °C. The phase composition and microstructures were evaluated, before and after the thermal processing, using XRD and SEM, respectively. Total porosity and bending strength of the samples were also tested. Proliferation and morphology of osteoblastic cells on samples were evaluated using MTT method. Limited growth of apatite crystals was observed by the thermal treatment in which the samples exhibited a crystal size of ~ 150 nm at heating temperature of 1250 º. Based on the results, the microwave irradiation led to a little change in phase composition of carbonated apatite and slight amount of β-TCP phase was found together with large amount of apatite. The sample irradiated at 1250 °C formed more dense material having bending strength value up to 130 % that of unheated sample. The in vitro cell studies showed that the microwave irradiated samples could provide adequate cell proliferation and attachment.

Abstract: The aim of this study was to evaluate the osteoconductive properties of synthetic porous hydroxyapatite prepared by low-temperature microwave processing OssaBase^® HA (SPHA) in comparison with biological apatite, non-sintered deproteinized bovine bone Bio-Oss^® (DBB). The materials were implanted into the bone sockets of the tibia of Beagle dogs for 3 and 6 months. The bone response to granules of the materials of similar sizes was compared. Histological analysis of the specimens with histomorphometry was performed at different times after in vivo implantation. Based on the histological analysis, the level of bone formation in the spaces between the implanted granules and through the interconnected pores of both implanted materials within a cortical region was significantly higher (bone area ingrowth 72–85%) than within a cancellous bone site (bone area ingrowth 16–28%) at three and six months after implantation. According to our study, the bioactive and osteoconductive properties (bone implant contact and bone area ingrown) of the synthetic porous hydroxyapatite are very high and comparable with the biological apatite, non-sintered deproteinized bovine bone. The favourable influence of the high specific surface area and carbonate content of the synthetic, porous hydroxyapatite on bone formation was confirmed.

Abstract: The objective of this study was to investigate the bone repair of carbonate apatite (cHA) in comparison to hydroxyapatite (HA, control group) on osseous repair of rabbit’s tibia. Spheres (400-500 µm) of both materials were synthesized under 37°C (cHA) and 90°C (HA) and were not sinterized. Ethics Commission on Teaching and Research in Animals approved this project (CEPA/NAL 208/10). Six White New Zealand rabbits were submitted to general anesthesia and one perforation (2mm) was made in each tibia for implantation of cHA (left tibia) and HA spheres (right tibia). After 4 weeks all animals were killed and one fragment of each tibia with the biomaterial was collected with a total of 6 bone blocks for each group. Five bone blocks of each group were demineralized and 5-µm thick semi-serial sections were stain with Hematoxillin and Eosin and Trichromic of Masson for histological analysis and two fragments were collected and embedded in a methacrylate-based resin and cut into slices with ~30 µm and were analyzed by light microscopy (bright field and polarized microscopy). Both groups didn’t show the spheres after 4 weeks, new-formed bone was observed from the periphery toward to the center of the surgical defect, which was even filled with connective tissue. Both materials are biocompatible, promote osteoconduction and showed to be resorbable.

Abstract: The objective of this study was to investigate the in vitro and in vivo biological responses to carbonate apatite (cHA) in comparison to hydroxyapatite (HA). Spheres (400<ø>500 μm) of both materials were synthesized under 5°C (cHA) and 90°C (HA) and not sintered. The in vitro cytocompatibility was determined by the XTT assay, according to ISO 10993-5:2009, after exposure of MC3T3-E1 cells to the materials extracts. Ethics Commission on Teaching and Research in Animals approved this project (CEPA/NAL 193/10) and, subsequently, the biomaterials were grafted in the subcutaneous tissues of mice (n=15). After 1 and 3 weeks, five animals of each group were killed for samples removal containing biomaterials and surrounding tissues for histological examination. Semi-serial (5-μm thick) sections were cut and stained with Hematoxylin and Eosin (HE) and the presence of inflammatory infiltrates and biomaterials resorption were evaluated. The experimental group of 3 weeks didn’t show the presence of spheres of both biomaterials and few spheres were observed after 1 week. Histological analysis showed the granulation tissue around the biomaterials with the presence of multinucleated giant cells. After 3 weeks it was observed the presence of fibrous tissue around biomaterials and few inflammatory cells. No signals of tissue necrosis were observed in both groups in all experimental studied periods. Nanostructured carbonate apatite spheres are cytocompatible, biocompatible and present initial biosorption on the subcutaneous comparable to stoichiometric HA, indicating its suitability for further studies on regenerative medicine.

Abstract: This study investigated the osteoinductive potential of granules of stoichiometric hydroxyapatite (HA) and 0.5% zinc containing hydroxyapatite (ZnHA) in intramuscular (IM) site of rabbit’s abdomen. The biomaterials were both used in granular form, with 75% porosity and particle diameter between 450 and 500μm, sintered at 1100°C. Both materials performed adequately on a multiparametric in vitro cytocompatibility assay, indicating their suitability for in vivo testing. After approval by the Ethics Commission on Teaching and Research in Animals, fifteen rabbits were submitted to general anesthesia, incision and tissue dilatation, and a small site was created for HA (right incision) and ZnHA (left incision) intramuscular implantation. The animals were killed after 2, 4 and 12 weeks for biomaterials and surrounding tissues removal. Histological analysis after 2 weeks revealed the presence of granulation tissue surrounding biomaterials with multinucleated giant cells and no newly formed bone for both materials. After 4 weeks there was fibrous tissue involving the material and few inflammatory cells. Following 12 weeks it was observed the presence of connective tissue surrounding the biomaterial, cellularized enough for the two experimental groups, but it was not observed the presence of bone matrix associated with the biomaterials. We conclude that both biomaterials are cytocompatible and did not present the property of osseoinduction after 12 weeks of implantation.

Abstract: The objective of this study was to investigate the bone repair of carbonate apatite (cHA) in comparison to hydroxyapatite (HA, control group), on osseous repair of non-critical size defect in rat calvaria. Spheres (400<ø>500μm) of both materials were synthesized under 37°C (cHA) and 90°C (HA) and were not sintered. Fifteen rats Wistar were submitted to general anesthesia and two perforations (4mm each) were made, one in each parietal bone, for implantation of cHA (left side) and HA spheres (right side). After 1, 3 and 6 weeks, five animals of each group were killed and the two fragments with the biomaterial were collected from the calvaria. The bone blocks with biomaterial were demineralized and 5μm thick semi-serial sections were done for histological analysis. The experimental group of 6 weeks did not show the presence of spheres of both biomaterials and few spheres were observed after 1 and 3 weeks. Histological analysis showed the connective tissue repairing the surgical defect after 1 week and newly formed bone after 3 weeks of surgery. Thus, we concluded both materials are biocompatible, promote osteoconduction and in all studied periods the biomaterials showed to be resorbable.