Key Engineering Materials Vols. 493-494

Abstract: Biological apatite presented in bone and teeth of mammals contains various minerals; thus, it has lots of defects with nano-scale sizes in the crystal structure. We fabricated hydroxyapatite ceramics including bone minerals (bone HAp ceramics) as model materials to clarify the relationship between nano-defect structure and bioactivity of biological apatite. The X-ray diffraction (XRD) results indicated that crystalline phase of pure and bone HAp ceramics were of HAp single phase. Chemical compositions of pure and bone HAp did not change before and after firing at 1000 °C for 5 h. Microstructure observed by high-resolution transmission electron microscope (HR-TEM) indicated that bone HAp ceramics contained more defects and strains compared to pure HAp ceramics. Osteoblasts derived from rat bone marrow cells (RBMC) were seeded on both pure and bone HAp ceramics. The level of differentiation into osteoblasts was examined by determining the content of alkaline phosphatase (ALP) for initial/middle stage and osteocalcin (OC) for late stage. The ALP activity normalized for DNA content of osteoblasts cultured on the bone HAp ceramics was higher than that of pure HAp ceramics. The OC amount normalized for DNA content of bone HAp ceramics was the almost same as that of pure HAp ceramics. These results demonstrate that the bone HAp ceramics may promote the differentiation into osteoblast.

Abstract: Cell adhesion, proliferation and differentiation are important specific parameters to be evaluated on biocompatibility studies of candidate biomaterials for clinical applications. Several different methodologies have been employed to study, both qualitative and quantitatively, the direct interactions of ceramic materials with cultured mammal and human cells. However, while quantitatively evaluating cell density, viability and metabolic responses to test materials, several methodological challenges may arise, either by impairing the use of some widely applied techniques, or by generating false or conflicting results. In this work, we tested the inherent interference of different representative calcium phosphate ceramic surfaces (stoichiometric dense and porous hydroxyapatite (HA) and cation-substituted apatite tablets) on different tests for quantitative evaluation of osteoblast adhesion and metabolism, either based on direct cell counting after trypsinization, colorimetric assays (XTT, Neutral Red and Crystal Violet) and fluorescence microscopy. Cell adhesion estimation after trypsinization was highly dependent on the time of treatment, and the group with the highest level of estimated adhesion was inverted from 5 to 20 minutes of exposition to trypsin. Both dense and porous HA samples presented high levels of background adsorption of the Crystal Violet dye, impairing cell detection. HA surfaces also were able to adsorb high levels of fluorescent dyes (DAPI and phalloidin-TRITC), generating backgrounds which, in the case of porous HA, impaired cell detection and counting by image processing software (Image Pro Plus 6.0). We conclude that the choice for the most suitable method for cell detection and estimation is highly dependent on very specific characteristics of the studied material, and methodological adaptations on well established protocols must always be carefully taken on consideration.

Abstract: Carbonated apatite (CHA) is commonly considered a promising synthetic material for biomedical applications in orthopedic and dental surgery due to its biocompatibility, bioresorption and bioactivity. CHA5, CHA37 and CHA90 powders were synthesized from wet method and the DRX patterns showed that the crystallinity and particle size of CHA samples increased proportionally with the synthesis temperature. Powder extracts medium were obtained from each sample to interact with MC3T3-E1 osteoblastics cells. It was evaluated morphology, citotoxicity, pH and Ca²⁺ concentration. Citotoxicity assays showed high metabolic activity on all samples when compared to control. The polygonal shaped and the confluent monolayer observed in control cells progressively changed according to the crystallinity increase of samples. Cells under mitosis and spindle-like shaped where the main alterations observed. In addition the cell viability could be sensitive to the acid reactivity and crystallinity of carbonated apatite samples.

Abstract: Addition of hydroxyapatite (HAp) into polymeric matrices and use them as supporting material for tissue engineering has gained attraction since hydroxyapatite (HAp) type crystals are the main inorganic components of the bone structure. In this study, composite films were prepared from chitosan (C) and gelatin (G) with addition of HAp, and the surface characterization and cell affinity properties were examined. The hydrophilicities and morphologies of the films were investigated by contact angle measurements and by scanning electron microscopy (SEM), respectively. Cell affinities were studied by using human osteosarcoma (SaOs-2) cell line. Crosslinked and HAp containing films were found to be more hydrophilic than that of the others, while surface free energy (SFE) values were almost similar for all films. Cell proliferation and spreading of SaOs-2 cells were higher for the films with high gelatin content and no HAp; whereas for HAp containing composites higher cell affinities were obtained for the samples which have high chitosan content.

Abstract: This study presents a novel synthetic approach for the preparation of cell attachable microfibers in microfluidic system. The synthesis is performed by multiphase laminar flows with spontaneous formation of carbon dioxide bubbles resulting asymmetrically porous PU microfiber. The fabricated Janus microfiber provides two distinctive properties: one is porous region to promote the cellular adhesion and the other is nonporous region rendering mechanical strength of the scaffold. The Janus microfibers show dramatic improvement of cell adhesion, proliferation, and viability over a culture period. The Janus microfiber can be used for not only alternative 2D cell culture plate but also as novel 3D scaffold for tissue engineering wihtout any need of elegant surface modification for enhancing cell adhesions.

Abstract: The first generation of synthetic bone substitute materials, hydroxyapatite (or HA), was initially investigated as a “non self-hardening” biomaterial for remodeling of bone defects. CPBCs concepts were used as a platform to initiate a second generation of injectable, self-hardening cement. The variety of CPBC’s chemical composition leads to a better understanding of their mechanism of reaction and their proposed classification: acid-base, mono-component and hydrolysable. After hydration, mixing, and full chemical reaction, these cements have the ability to precipitate different end products (e.g. HA, calcium deficient apatite, carbonated apatite, brushite, etc.). In fact, the initial idea of having higher mechanical performance (>50 MPa in compression) for a bone void filler application was abandoned and has led to a greater focus on cement fast-hardening (<15 min), higher total porosity (>60%), extended performance of injectability (8 to 22 G), fast resorbability (< 2 years) and user-friendliness for the clinicians. A new CPBC combination (cement plus additives) has particularly improved rheological and biointegrity performance. A hybrid of CPBC-DBM (Demineralized Bone Matrix) has also added an osteoinductivity performance to the initial osteoconductive CPBC.This paper will propose a comparison of the chemical composition, reaction, and performance characteristics of major commercially available CPBC products. Furthermore, it will describe today’s surgeon’s CPBC needs as bone substitute materials for different clinical applications. Finally, we will discuss what we learned so far, how we can resolve several clinical impacts & product recall, and how we believe CPBC designers can meet development challenges, and users’ specific requirements.

Abstract: The paper presented here deals with rheological and hardening properties during the setting reaction, and density and compressive strength after the final setting of a figuline composite consisting of Ca₂KNa(PO₄)₂ and 2wt% medium gel strength gelatin. Compared to the composite with monomodal particle size distribution (d₅₀=7.18µm; span=3.9) and its properties during and after setting reaction, the goal of this work is to increase the resulting product compressive strength by mixing different particle sizes in order to obtain bi- and trimodal distributions. For the bimodal powder mixtures the ratio in diameter (d_course/d_small) was chosen with 7/1 and volume ratio d_course/d_small was 70/30%. For the trimodal powder mixtures the ratio in diameter (d_course/d_medium/d_small) was chosen with 70/7/1 and volume ratio d_course/d_medium/d_small was set to 44/28/28%.After establishing an adequate crushing and sieving process the tap density and powder density of each fraction was determined. Subsequently, the different particle sizes were mixed and the densities and the Hausner ratio were determined again. The mixtures show an increase in both densities especially the tap density increased significantly. Rheological investigations show that the graphs of storage and loss moduli of the multimodal powder mixtures respectively are similar. The characteristic setting times show a slight decrease compared with the monomodal composite but not significantly different data. When comparing the resulting compressive strength of cylindrical samples, which were stored direct after reaching the initial setting time under physiological conditions, the studies illustrated in all cases for the multimodal mixtures a significant increase in compressive strength and a higher density.

Abstract: Antibiotics release impregnated in HA granules, which were used in IBBC to prevent infection after total joint arthroplasty, was measured. For antibiotics, Flumarin, Vancomycin, Pansporin and Firstcin were used. Two models of antibiotics release were assumed; Model [I]: antibiotics release from surroundings of HA granules immediately after surgery and Model [II]: antibiotics release loaded on HA after antibiotics release from surroundings of HA granules as follows; (1) loading in normal pressure and (2) loading in reduced pressure. The amount of antibiotics loaded on HA is higher when loading is conducted under reduced pressure than that under normal pressure. Firstcin showed the highest loaded amount and the most desirable sustained release pattern. The antibiotics release from HA are varid depending on the antibiotics used.

Abstract: To improve the fixation of bone and bone cement in THA we used Interface Bioactive Bone Cement technique (IBBC) since 1982. For prostheses 28 mm-alumina head with polyethylene socket (Kyocera Co.) were used. In IBBC not-resorbable pure crystalline HA granules (0.3– 0.5 mm) were smeared on the bone surface in one to three layers just before packing bone cement. Group 1: IBBC was used in six joints in 1982. Group 2: In 25 patients, conventional bone cement technique (non-IBBC) in the one hip (in 1985 to 1986) and IBBC technique in the other hip (in 1986) were performed in the same patients. Group 3: In 65 patients (71 joints) IBBC were performed. Neither radiolucent line nor osteolysis appeared in all case in group 1. In group 2 in Non-IBBC the appearance rate of radiolucent line and osteolysis were rather high, however in IBBC they were extremely low. In Non-IBBC loosening were seen in 5 joints in the acetabulum and in 2 joints in the femur. However, in IBBC there was no loosening. In group 3 the radiolucent line and the osteolysis appeared extremely low. There was no loosening. In Non-IBBC the appearance rate of radiolucent line and the osteolysis was very high and the rate of loosening was 20%. However, in IBBC, radiolucent line and osteolysis will be prevented eternally.

Abstract: We have developed novel hydroxyapatite (HAp) cement, “chelate-setting HAp cement” on the basis of chelate bonding of inositol hexaphosphate (IP6). In order to create the cement with enhanced bioactivity, we used a silicon-substituted hydroxyapatite (Si-HAp) as a starting material instead of pure HAp powder. The Si-HAp powders were prepared via an aqueous precipitation reaction and surface-modified with IP6 (IP6-Si-HAp). The Si-HAp were synthesized with desired Si contents (0, 0.4, 0.8, 1.6, and 2.4 mass%). Regardless of the amount of Si, the XRD patterns and FT-IR spectra of the powders were similar to each other. Additionally, chemical composition of Si-HAp powders were the almost same as the nominal one. The substitution of Si resulted in a decrease in the a-axis and increase in the c-axis of unit cell of HAp. While, the compressive strength of IP6-Si-HAp cements was higher than that of Si-HAp cements. These results suggest that surface-modification of any powders with IP6 is effective for enhancement of mechanical property. Comparison of mechanical property between HAp and Si-HAp cement specimens revealed that the substitution of lower levels of Si into0 HAp lattice did not greatly influence compressive strength. However, the substitution of high dose of Si (over 2.4 mass%) reduced the compressive strength. Therefore, to fabricate the chelate-setting cements with enhanced bioactivity, it is necessary to control the amount of Si.