Hydroxyapatite Ceramic Surfaces Research Articles

Efficient quantification of primary amine functions grafted onto apatite ceramics by using two UV-Vis spectrophotometric methods

Enhancement of osteogenic properties of bone substitutes is best achieved by the covalent attachment of appropriate biomolecules, e.g. proteins and peptides, on amino-functionalized implant materials. Measurement of the free amino surface density of these materials is mandatory before derivatization. Two photometric methods were developed to assay the free amino groups of organosilane molecules covalently grafted onto hydroxyapatite and silicon-substituted hydroxyapatite ceramic surfaces. Reaction of amino-derivatized apatites with ninhydrin led to the formation of Ruhemann's purple quantified by the absorbance at 586 nm. Alternatively, ceramics were reacted with Fmoc-derivatized amino acids in the presence of piperidine, leading to the dibenzofulvene–piperidine adduct measured at 301 nm. Both methods allow the detection of free amino groups down to 1.0 × 10−8 mol g−1 of apatite-ceramics and, since they are conducted under non-acid conditions, preserve the acid-sensitive structure of hydroxyapatite.

Apatite Formation on Titanium Containing Apatite

Some titanium containing apatite (TMA) ceramics prepared by wet synthesis was superior to Hydroxyapatite (HAp) ceramics of mechanical property and cutting performance. In this study, we investigated chemical reaction of TMA in a body environment. Each TMA or commercial Hydroxyapatite powder (HAp) was formed to circular pellets by uniaxial pressing. The specimens were heated in muffle furnace by conventional sintering process. Some TMA specimens were dispensed by alkali and heat treatment. After the treatments, the specimens were soaked in simulated body fluid (SBF: Kokubo’s solution) at pH 7.25 at 37.0°C up to 7 days. Before and after soaking in SBF, the surfaces of the samples were characterized by XRD, SEM, and ICP. After soaking in SBF for 7days, some particles were precipitated on the surface of HAp ceramics and TMA ceramics with alkali and heat treatment. Ti-containing in the crystal structure of HAp affected apatite formation in the body environment.

소 뼈로부터 제조한 다공형 하이드록시아파타이트 세라믹스의 생체친화 특성

소뼈를 하소하여 천연 하이드록시아파타이트 분말을 얻은 다음, <TEX>$1100^{\circ}C$</TEX> 및 <TEX>$1200^{\circ}C$</TEX>로 상압 소결하여 기공율이 각각 35 % 및 18 %인 다공성 하이드록시아파타이트를 제조한 후 이들 시편의 생체친화성을 평가하였다. 골아세포의 성장속도과 세포독성의 유무에 따라 평가되는 생체친화성은 MG-63 세포의 부착능과 분화되어 증식되는 세포의 수로 측정하였다. in vitro에서 세포 반응을 관찰한 결과 소뼈로부터 제조된 하이드록시아파타이트 시편은 독성이 없고 생체 친화성이 좋은 시편이었다. 세포배양 1일 후부터 하이드록시아파타이트 표면에 세포가 부착되었고, 세포배양 후 3일 후에는 핵 주위로 세포질이 퍼져나갔으며, 5일 후에는 부착된 세포의 수가 크게 증가하였다. 배양 초기에는 밀도에 관계 없이 세포의 부착능이 유사하였지만, 세포 배양 시간이 증가하면서 기공율이 높은 시편에서 세포의 부착 및 성장이 더 활발하게 진행되었다. Natural hydroxyapatite powder was obtained from the calcination of bovine bones and its porous compacts were fabricated by pressureless sintering at 1100 and <TEX>$1200^{\circ}C$</TEX> for 1h. To evaluate and compare their biocompatibility with porosity, we investigated the support of osteoblast cells growth and cytotoxicity using the MG-63 cell line model in vitro. Sintered hydroxyapatite ceramics have a porous microstructure with a relative density of 65 % at <TEX>$1100^{\circ}C$</TEX> and 82 % at <TEX>$1200^{\circ}C$</TEX>. Cells adherence to the surface of hydroxyapatite ceramics was observed in a day after the cell culture, and the spreading of cytoplasm around the nucleus was shown after 3 day cell culture. Most of cells were extended to the surface of hydroxyapatite through the wide area. Cell viability was nearly the same till 3 days culturing. But the rate of cell growth is higher in the specimen sintered at <TEX>$1100^{\circ}C$</TEX> than that of <TEX>$1200^{\circ}C$</TEX>. It indicates that the porosity is an important factor to enhance the cell viability in the porous hydroxyapatite ceramics derived from bovine bones.

Immobilisation of yeast cells on the surface of hydroxyapatite ceramics

Hydroxyapatite (HAP) ceramics was tested for the first time for the possibility of being used as a new carrier for the immobilisation of yeast cells that are both model organisms for eukaryotic cell investigations and producers, which is important in classical and modern biotechnological processes. It was shown that under typical immobilisation conditions yeast (Saccharomyces cerevisiae) has no affinity to HAP ceramics. A novel method for yeast immobilisation was developed. This new method includes the joint incubation of a carrier with the cells, the sedimentation and adhesion of cells on the carrier and the dehydration of obtained preparations. It was shown that the sedimentation and adhesion stages strongly depend on the value of the electron work function of the HAP ceramics surface. An increase in this value, which corresponds to a higher negative charge on the ceramics surface, correlates with higher cell immobilisation. The dehydration stage strongly and stably fixes cells on the ceramics. Experiments with yeast cells immobilised on HAP ceramics have shown the efficiency of using these preparations in biotechnologies linked with the purification of wastewaters from heavy metals.

Difference in Bioactivity, Initial Cell Attachment and Cell Morphology Observed on the Surface of Hydroxyapatite Ceramics with Controlled Orientation

Hydroxyapatite ceramics has been demonstrated to be an appropriate material for biomedical applications owing to its bioactivity and high biocompatibility. It has an anisotropic crystal structure that belongs to the hexagonal system, and two types of crystal planes mainly appear on its crystal, which are a-plane and c-plane. Since these two crystal planes are very different in atomic elements, numbers and arrangements, they exhibit different nature (anisotropy). For this reason, it is said that crystal orientation might be intensifying its bioactivity and biocompatibility. However, the differences in biological features on these two crystal planes are not fully clarified yet. In this study, we have conducted an assessment to reveal anisotropic biological features of hydroxyapatite by using hydroxyapatite ceramics with controlled orientation fabricated by slip casting under a magnetic field. Tanase et al. have recently reported the difference in bioactivity on the two crystal planes by immersing crystal oriented hydroxyapatite ceramics into the simulated body fluid and found that c-plane oriented hydroxyapatite ceramics formed a precipitate layer earlier and thicker on its surface compared to a-plane oriented one. We first carried out Welch’s t-test on the difference in the thickness of the precipitate layer, reported previously to reveal the difference in bioactivity. Secondly, we conducted an osteoblast culture experiment on hydroxyapatite ceramics with controlled orientation to reveal the difference in initial cell attachment and cell morphology on the two crystal planes of hydroxyapatite using optical microscope observations. In the former case, the results of the Welch’s t-test indicated that the thickness of the precipitate significantly differed on each crystal oriented hydroxyapatite ceramics (P < 0:05). In the latter case, initial cell attachment seemed to be better on the a-plane oriented hydroxyapatite ceramics and also the morphology of the osteoblasts seemed to be rounded on the a-plane oriented hydroxyapatite ceramics compared to the c-plane oriented one. [doi:10.2320/matertrans.MBW200836]

Enhanced bone morphogenetic protein‐2 performance on hydroxyapatite ceramic surfaces

The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces.

Formation of a FGF-2 and calcium phosphate composite layer on a hydroxyapatite ceramic for promoting bone formation

Fibroblast growth factor-2 (FGF-2) was immobilized on a hydroxyapatite (HAP) ceramic in supersaturated calcium phosphate solution prepared using solutions corresponding to clinically approved infusion fluids. To avoid the risk of FGF-2 denaturation, FGF-2 immobilization was carried out at 25 °C. FGF-2 was successfully immobilized on HAP ceramic surfaces by deposition with calcium phosphate to form a FGF-2 and calcium phosphate composite layer. A maximum of 2.72 ± 0.01 µg cm−2 of FGF-2 was immobilized in the composite layer formed on the HAP ceramic under the optimum condition. A FGF-2-immobilized HAP ceramic is likely to have the ability to release a sufficient amount of FGF-2 to promote bone formation. FGF-2 released from a FGF-2-immobilized HAP ceramic maintained its biological activity, since the proliferation of fibroblastic NIH3T3 was promoted. Therefore, the FGF-2-immobilized HAP ceramic is expected to be a useful material for promoting new bone formation.

Chemical Functionalization of a Hydroxyapatite Based Bone Replacement Material for the Immobilization of Proteins

Hydroxyapatite (HAP) has a great potential as a bone replacement material because of its similarity to the crystal structure of inorganic matrix of bone. Several years ago we showed that porous hydroxyapatite ceramics (Endobon, Merck) can be covalently modified for the immobilization of proteins [Jennissen, H.P. (1999) PCT Patent WO9926674A2] opening the possibility for the immobilization of growth factors and morphogens. Application of this technique to the immobilization of recombinant human bone morphogenetic protein 2 (rhBMP-2) on hydroxyapatite would be of high medical interest, because of the decisive role of bone morphogenetic proteins in bone development and osteogenesis. In this paper it will be shown that the model protein ubiquitin and rhBMP-2 can be immobilized non-covalently and covalently after chemical modification of a hydroxyapatite ceramic surface (Bio-Oss J Cancellous Block, Geistlich, = HAP-wafers). It could be shown that only small amounts of protein are adsorbed to non-functionalized HAP surfaces (control). In contrast ubiquitin and rhBMP-2 can be very efficiently immobilized non-covalently (up to 2.4mg protein/g HAP) and covalently (up to 9.7mg protein/g HAP) on porous HAPwafers. In desorption experiments it is shown that the bound rhBMP-2 is slowly released making such surfaces applicable as long term drug delivery devices for enhancing bone growth and osteointegration of implant materials.

Effect of Phase Components on Apatite Formation on the Surface of Ca-P Bioceramics

Dense hydroxyapatite (HA) and hydroxyapatite/tricalcium (HA/TCP) were immersed in Fast calcified solution (FCS) at 37°C in constant temperature culture box for 7 days. It was observed that the phase components of dense ceramics affected the composition, crystallinity and phase components of formed apatites on the surface of ceramics. It is a mixture of hydroxyapatite with a strong preferential crystallographic direction of 002 and octacalcium phosphate (OCP) on the surface of HA/TCP ceramics, whereas only an HA phase was detected on the surface of HA ceramics.

Conformation of self-assembled organic layer on the electrically polarized hydroxyapatite ceramics in nonaqueous solution

The conformation of organic compound deposited on hydroxyapatite (HAp) ceramics was manipulated by their surface charge induced by electrical polarization. The sintered HAp ceramics were electrically polarized in a d.c. field at 400 °C. The organic layers on the HAp ceramics were deposited from a toluene solution of phenyltrichlorosilane (PTCS). The results of contact angle measurements and IR spectroscopy clarified the organic layer containing the fragments of PTCS. The amount of the obtained layer on the positively charged surface was larger than those on the negatively charged and nonpolarized surfaces. The thickness of the layer on the nonpolarized surfaces was thinner than those on the polarized surfaces, based on the depth profiles of Ca2p and Si2p. Although the deposition prepared on the nonpolarized HAp surfaces from the PTCS solution was supposed to be a self-assembled layer including of phenyl–Si bond, the layers on the polarized HAp were aggregated irregularly. Consequently, the HAp ceramic surface charge induced by the polarization altered the electrostatic interaction to the organic compounds in nonaqueous system.

Extended bioactivity in the proximity of hydroxyapatite ceramic surfaces induced by polarization charges.

Surface charges of electrically polarized hydroxyapatite (HAp) ceramics were demonstrated to enhance osteoconductivity in the wide gap of the canine bone whereas the bone formation processes varied according to the charge polarity. Cell reactions in the vicinity of the charged surfaces of HAp ceramics were not phagocytic absorption but rather bone formation by stimulated osteoblasts surrounding newly formed bone 7 days after implantation. The bone formation in direct contact with negatively charged ceramic surfaces suggests that the negative charges enhanced the osteobonding ability of the HAp ceramics. In the vicinity of the positively charged HAp surface, the formation of bones derived from osteoid tissues entirely occupied the 0.2-0.3-mm gaps between the HAp and the bone at 7 days. Surface charges induced by electrical polarization significantly cooperated with the innate bioactivity of HAp and reconstructed the wide bone defects more promptly than did the nonpolarized HAp ceramic surface.

Enhanced Mineralization on Electrically Polarized Hydroxyapatite Ceramics in Culture Medium.

Growth enhancement of the bone-like layer on electrically polarized hydroxyapatite was investigated. Dense hydroxyapatite ceramics were electrically polarized in a dc field. For estimation of the bone-like grown layer on the polarized hydroxyapatite ceramic surfaces, polarized specimens were immersed not only in a simulated body fluid (SBF) with the inorganic ion-concentrations equal to those of human blood plasma, but also in minimum essential medium (MEM), and in MEM supplemented with 10% foetal bovine serum. After several days of immersion, the grown crystal layers were observed by scanning electron microscopy, and analyzed by X-ray diffraction and IR spectroscopy. As a result, the bone-like hydroxyapatite crystals grew rapidly on the negatively polarized surface, while the growth was restricted on the positively polarized surface both in SBF and MEM. The morphology of the grown layers on the polarized surface was different among the immersion fluids. The growth enhancement of the bone-like layer on the polarized HAp was demonstrated in multiple ions based on inorganic and organic groups. It was considered that the charged surfaces of the polarized HAp ceramics accelerated the growth of the bone-like layer on the negatively polarized surface, but was inhibited on the positively polarized surface.

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

Modification of Assembled Structures of Steroids Adsorbed on Apatite Ceramic Surface by Electrical Poling

AbstractAggregate structures of steroids adsorbed on electrically-poled hydroxyapatite (HAp) ceramics were investigated by X-ray diffractometry (XRD) and scanning electron microscopy. Effectivity of the poled HAp on modification of the steroid assembly modes was discussed by the arrangement estimations of the steroid molecules. The dense HAp ceramics were obtained from the pelletized HAp powder precipitated from calcium hydroxide suspension and phosphoric acid using conventional sintering at 1250°C under the saturated water vapor pressure. The HAp ceramic blocks were poled in a DC electrical field of 1.0 kVcm-1 with being heated at 300°C. The HAp ceramics were immersed in ethanol solution of steroids at 37°C. The steroid crystals were overgrown onto the HAp substrates by recrystallization method. In the case of cholesterol, the tabular crystals of cholesterol monohydrate and the needle-like crystals of anhydrate were overgrown on all of the HAp ceramic surfaces. The thickness of the cholesterol layers on the positively (p) poled HAp surfaces was considerably less than those of the negatively (n) poled and non-poled (0) surfaces. The remanent chaige of the HAp ceramics altered the morphology of vitamin D3 crystals deposited on the HAp surfaces. It was revealed that the electrically-poled HAp ceramic surfaces changed the aggregate structures and the crystal growth rates of steroids.

Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics

To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 122–129, 1997.

Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics.

To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding.

Formation of a bone apatite-like layer on the surface of porous hydroxyapatite ceramics

Phosphoric acid solution was used to react with commercial hydroxyapatite (HA) powders to demonstrate the possibility of converting HA to non-stoichiometric apatite and thus to treat porous HA ceramics, to form a thin bone-apatite like layer on the HA ceramic surface. Such a carbonate-containing non-stoichiometric apatite ‘bioceramic’ would be more efficient in bone bonding or bone formation, due to its analogy to natural bone apatite.

Surface properties of hydroxyapatite ceramic as new percutaneous material in skin tissue

Hydroxyapatite ceramic (HAC) was studied for utilization as a percutaneous device to prevent exit-site and tunnel infection along peritoneal dialysis catheters. As a result, it was found that HAC had good compatibility with skin tissue compared with silicone rubber and glassy carbon. In the present study, the surface properties of HAC after long-term implantation in skin of dogs was evaluated by scanning electron microscopic observation, solubility and bending tests. At 12 weeks after implantation, the surface of HAC was eroded severely by body fluid or cells, and the grain boundaries were clearly relieved. Ingrowth and adhesion of collagen into the etched ditch of HAC were observed. At 12 months, the eroded surface had been tightly covered and adhered to by a collagenous layer in spite of ripping off the surrounding fibrous capsule. In solubility tests, HAC dissolved at the rate of 0.23 mg cm−2 HAC surface area per year. On the other hand, the bending strength decreased by about 10% at 4 weeks and scarcely decreased after that time. From these results, it was confirmed that the solubility of HAC produced tight contact and strong adhesion with skin tissue to prevent bacterial infection, and HAC was practical for a permanent percutaneous device.

Tensile strength of the interface between hydroxyapatite and bone.

Tensile strength of the interface between hydroxyapatite (HA) and bone was tested. Scanning electron microscopy was used to observe the tensile failure mode and the morphological change of hydroxyapatite ceramic surface in bone. The porosity of hydroxyapatite is 14% and pore size less than 2 microns. After 2 weeks of implantation, the tensile strength of the interface is 0.72 MPa. After 4, 8, and 16 weeks, the average tensile strength stayed at 1.5 MPa. SEM showed that tensile failure occurred at the HA-bone interface at the second week, but after 4 weeks, the failure occurred between HA particles within the bulk, and not at the HA-bone interface. Calcified tissue was directly deposited on the HA ceramic surface and exits also in the micropores. Near the interface, sintered necks among HA ceramic particles were subjected to biodegradation.