Key Engineering Materials Vols. 493-494

Abstract: Porous granules were obtained through a route using a mixture of calcium phosphate powder with sodium alginate solution. The sintered β-TCP and β-TCMP granules with diameters ranging from 250 µm to 500 µm were implanted into dental alveoli of 30 rats (Rattus norvegicus). The animals were divided in three groups: group I (control, no treatment), group II (β-TCP) and group III (β-TCMP); the sacrifices occurred at 7 and 21 days (n=5/group/period). Histological and histomorphometric analysis were performed to observe and measure connective tissue, bone neoformation and biomaterial areas. Most animals showed acute inflammatory response with many neutrophil granulocytes and foreign body giant multinucleated cells associated to both biomaterials, at 7 and 21 days. The utilization of sodium alginate as additive in the porous granules might explain these results. Fourier-transformed infrared spectroscopy (FTIR) pointed out residue in the granules surface that could exacerbate the inflammatory response. Additional studies are in course to confirm such hypothesis.

Abstract: Orthopedics surgeries frequently are open surgeries, but the improvement of the specific instrumentation and the use of bioresorbable polymerics implants for regeneration of bone fractures are contributing to the development of noninvasive techniques such as an injectable bone substitute. These injectable materials are composites, formed by a particulate ceramic phase and a polymeric phase, and have the advantages of combining bioactivity and the ability to control degradation and some mechanical properties. In addition, microparticles present flexibility to fill several types of defects with closer packing and allow new bone growth and vascularization through the interconnected pores formed by the spaces between them. Another advantage of the particulate materials is that they have the potential to incorporate drugs such as antibiotics that can be applied in situ for treatment or prevention of bone infection, which is important because the poor circulation of blood in the osseous tissues makes necessary large amounts of these drugs to guarantee that an adequate dose reaches the affected site. This work evaluates the release potential of gentamicin from BCP spherical microparticles to be used in osseous injectable implants. The particles present a smooth geometry to prevent inflammatory reactions frequently caused by an irregular morphology, and their compositions offer a combination of biodegradability and stability. Microparticles with diameters between 150-425µm, were obtained by a method based on the immiscibility of liquids. To encapsulate the antibiotic, the spheres were immersed in a gentamicin solution, and after 24h they were separated and dried. The evaluation of the gentamicin release from the microspheres was carried out at 37°C in PBS, and the release medium was collected at predetermined time intervals for measurement of the amount released. This work demonstrates that these microspheres can find potential application in bone repair and regeneration.

Abstract: In situ viscous mass foaming with ammonium bicarbonate (NH₄HCO₃) as foaming agent was used for the preparation of porous hydroxyapatite ceramics. Three different particle fractions (100-300 μm, 300-500 μm, 500-1000 μm) of NH₄HCO₃ were used in order to evaluate the particle size effect on the ceramic microstructure. Foamed and dried green bodies were sintered at 1150°C. The porosity and microstructure of ceramics were determined with Archimedes method and scanning electron microscopy. As the NH₄HCO₃ particle fraction was decrease from 100-300 μm to 500-1000 μm, the open porosity increased from 42 to 54 %. In situ viscous mass foaming leads to the interconnected pore channels with wide pore size range (10-600 μm). In the present work cytotoxicity of obtained porous HA ceramics and fibroblast cell viability was investigated.

Abstract: Hydroxyapatite (HA) is a particularly attractive material for bone and tooth implants since it does not only closely resemble human tooth and bone mineral but it has also biologically proven to be compatible with these tissues. The applications of pure HA are restricted to non load bearing implants due to the poor mechanical properties of HA. Biomaterials of synthetic HA are highly reliable but the synthesis of HA is often complicate and expensive. Bioceramics of naturally derived biological apatites are more economic. Aim of the present work is to introduce sheep teeth dentine HA material as an alternative source of bioactive biomaterials for grafting purposes. The work was started with such a way that extracted sheep teeth were cleaned. The teeth were calcinated at 850°C in air. It was seen that enamel matter was easily separated from dentine after calcination. The collected dentine parts were crushed and ball milled. The powder was pressed between hardened steel dies to produce samples suitable for compression and microhardness tests. The obtained powder compacts were sintered at different temperatures, specifically 1000, 1100, 1200, and 1300°C in air. Results of microhardness and compression strength (along with the statistical analysis of these results) as well as those of SEM and XRD analyses are presented. In the literature, there is very little information about the mechanical properties of dentine and enamel matter derived from sheep, bovine and human. The highest compression strength value in the present study was measured around 146 MPa (from human dentine derived HA the highest value was almost 60 MPa after sintering at 1300°C). The best microhardness in the present study was found as nearly 125 HV. The results of this study showed that the HA material produced from sheep tooth dentine can be qualified as a promising source of HA needed to produce bioactive ceramics.

Abstract: There are thousands of land snail species, ranging in size from 1 mm to the Giant African Snail growing up to a foot long. Two species, known as escargot, helix aspersa and helix pomatia, are commercially important. Helix pomatia is abundant in Turkey. Those snails are exported usually without shells. Shells are damped to trash sites or used as substitute food for animals. The shell is rich in calcium carbonate and some other minor minerals. Thus, snails’ shells can be used as a source for bioceramic production. So far, in the literature there are lot of papers about converting calcite and aragonite structures to hydroxyapatite (HA), like corals, sea shells, sea urchin and other sea creatures. However, there is very limited information about converting land snail shells to HA and other bioceramic phases. The aim of this work was to produce various phases of bioceramic materials from land snails’ shells which are left as a residue waste after their export procedures. Empty local land snails’ shells (helix pomatia) were collected in Istanbul. They were washed, dried, crushed and ball milled until a powder of 100 µm particles size was obtained. Raw powders were stirred at 80°C for 15 min on a hotplate. A second part of the raw powder was stirred with an ultrasonic stirrer at 80°C for 15 min in an ultrasonic equipment. Equivalent amount of H₃PO₄ was added drop by drop into the solution. The reaction lasted for 8h. Then, to evaporate the liquid part, the mixtures were put into an incubator at 100°C for 24 h and the resultant dried sediments were collected. The produced powders were analyzed with X-ray diffraction, IR and scanning electron microscope (SEM). The experimental results confirmed the formation of various Ca-phosphates, specifically monetite, fluorapatite and some other minor calcium phosphate phases. Bioceramic production from land snail is a reliable and economic way comparing to other tedious methods of producing synthetic HA and other various bioceramics phases.

Abstract: Carbonated apatite, the basic mineral component in human hard tissues and an important bioceramic material, has been extensively studied. However, its atomic arrangements in apatite crystal structure and its experimental characterization are still not lack of debating. We analyzed infrared (IR) vibrational spectroscopy for carbonated apatite determinations, by comparatively studying the IR spectra of hydroxyapatite and of surface carbonate absorption, biological apatites (human enamel, human cortical bone, and two animal bones) and carbonated apatite. The carbonated apatite samples were sythesized by various methods, including precipitation method, hydrothermal reaction and solid-gas reaction at high temperature. The comparative study indicates that the bands at ~880 cm^-1, ~1413 cm^-1, and ~1450 cm^-1 should not be used to identify carbonated apatite since they may result from carbonate absorption on surfaces of apatite crystals or separated carbonate phase present with apatite crystals. The IR characteristic bands of carbonate substitution in apatites should be: ν₃ at ~1465 cm^-1 for type-B (CO₃ substituting for PO₄) and ν₃ band at ~1546 cm^-1 for type A (CO₃ substituting for OH). These signature IR bands are further confirmed by the ab initio simulations.

Abstract: Hydroxyapatite (HA) is one of the most employed materials for bone therapy due to its structural similarity with bone, its biocompatibility and physicochemical properties. Additionally, HA performance may be improved by ionic substitution of calcium with divalent bioactive metallic cations such as zinc. In this context, zinc incorporation into HA have been well studied, in spite of conflicting results regarding its biocompatibility: while previous reports on in vitro cytocompatibility have described 5% zinc containing HA (ZnHA) as slightly cytotoxic, this material presented an excellent response on in vivo studies. In order to bring more information on ZnHA biocompatibility, we performed a multiparametric assay evaluating sequentially on the same cells three different viability parameters: mitochondrial activity (XTT), membrane integrity (Neutral Red) and cell density (Crystal Violet Dye Exclusion test). Additionally, we intended to complement the existing data on ZnHA in vivo performance, by assessing its ability to affect the arrangement of collagen fibers on the grafted area, an important indicative of bone maturation. MC3T3-E1 cells were exposed to 24-hours extracts of ZnHA or stoichiometric HA on culture medium (DMEM) and cell viability was assayed. ZnHA was very cytocompatible, since the levels of viable cells on all 3 tests were similar to the HA and polystyrene (negative control) extracts, but significantly higher than cells treated with 4% phenol (positive control). For the in vivo studies, critical size defects in rats calvaria were filled with HA or ZnHA granules. The histological evaluation after 30 and 180 days revealed an increase along time. Event tough ZnHA is cytocompatible the presence of Zn was unable to alter the interaction between collagen fibers and the mineral bone phase, as compared to stoichiometric HA.

Abstract: Hybrid composites with chitosan (CHI), silk fibroin (SF) and hydroxyapatite (HA) are biocompatible and attractive for bone engineering applications. The objective of this work was to evaluate in vitro cells behavior in contact with CHI-SF-HA scaffolds. The scaffolds were produced from a chitosan solution (2%wt) with SF (1%wt) and HA powders (1%wt) in acetic acid. This solution was molded, frozen and the scaffolds were freeze-dried, crosslinked, and then, submitted to in vitro tests with STRO+1A, MC3T3-E1 and SaOS₂ cells under static conditions for 7, 14 and 21 days. The scaffolds were characterized through X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Cell viability and activity was assessed by MTT reduction and alkaline phosphate (ALP) activity detection. XRD patterns showed characteristic peaks at 8.8º, 20.3º and 24.6º (corresponding to SF) and peaks at 31.8º, 32.2º and 32.9º (corresponding to HA). The FTIR presented characteristic bands of the amide groups (SF) and of the phosphate and carbonate groups (HA). The EDS showed the presence of the C, O, N, P and Ca elements. SEM analyses showed the scaffold morphology, and indicated cell growth across the surface of the sample. The STRO+1A and MC3T3-E1 cells presented the best cell adhesion. MTT assay showed an increase of the cell number with time and the 21 days analyses showed the best proliferation for SaOS₂ cells (p<0.01), STRO+1A cells (p<0.04) and MC3T3-E1 cells, respectively. The ALP activity was higher at 21 days for all cells types and the SaOS2 cells presented the best results in all analyses (p<0.01). Molecular studies are under progress to evaluate more deeply the biocompatibility of these scaffolds. Moreover, dynamic studies in bioreactor are under progress to improve cell colonization inside scaffolds.

Abstract: Bone and dentin consist of hydroxyapatite, collagen and body fluid. From biological points of view, we have been focusing on HAp and collagen materials for bone regeneration. The aim of this study is to estimate the appearance of multinuclear giant cells for non-organic (functionally graded HAp: fg-HAp) and organic materials (demineralized dentin matrix: DDM), histologically. The fg-HAp ceramic: Biomimetic fg-HAp was designed by using the partial dissolution-precipitation methods. The fg-HAp with micro-pores of 10-160 nm had larger specific surface areas (30-40 m²･g^-1) than the synthetic HAp. Acid- insoluble dentin matrix (DDM): Human teeth were crushed under the cooling, completely demineralized in 0.026N HNO₃ solution, and dried. The materials were implanted into the subcutaneous tissues (Wistar rats, 4 week-old, male), and removed at 1 and 4 weeks after the operations. Multinucleated giant cells were counted in the H-E sections. Giant cells predominantly appeared on the biodegradable micro-crystals at 1 week. The number of giant cells was more numerous in fg-HAp than in DDM. There was a significant difference in the cell number between fg-HAp and DDM. The absorption mechanism of fg-HAp should be predominantly cellular phagocytosis, while that of DDM might be predominantly enzymatic digestion. These data support the hypothesis that the biological HAp crystals may function as mineral signal in the recruitment and differentiation of multinucleated giant cells.

Abstract: The anti-tumor activity of hydroxyapatite (HAp) cements, which had been developed using a novel setting mechanism termed chelate bonding, against the human osteosarcoma cell line (HOS) and rat bone marrow stromal cells (BMSC) was examined. We aimed to understand the mechanism of the anti-tumor activity of the cement, thereby facilitating improvement of its biological activity. HAp powders were surface-modified with three different concentrations of inositol hexaphosphate (IP6), which were then used to fabricate three different IP6-HAp cements. The amount of IP6 that was bound to the HAp powder, and the amount that was released from the HAp cement, was measured for each sample. Approximately 1/200 to 1/1600 of the bound IP6 was released into the culture medium by day 4. Surface-modification of HAp with high concentration of IP6 inhibits the proliferation of both HOS cells and BMSCs, and appears to induce their apoptotic cell death. HOS cells were slightly more sensitive to IP6 than BMSCs. Thus, novel, chelate-bonded HAp cements are a candidate bone substitution material that exhibit anti-tumor activity.