Effects of dissolved calcium and phosphorous on osteoblast responses.

The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization.

Characterization and dissolution behavior of sputtered calcium phosphate coatings after different postdeposition heat treatment temperatures.

There is a lack of correlation between specific properties of hydroxyapatite coating surfaces, osseointegration processes, and implant success. The aim of this study was to evaluate the relationship between well-characterized structural and chemical properties of radio-frequency sputtered calcium phosphate (CaP) coatings and their dissolution behavior. Sputtered CaP coatings were evaluated as-sputtered (non-heat treated) or after 1 hour of postsputter heat treatments at 400 degrees C or 600 degrees C. All coatings were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and contact angle measurement. The dissolution behavior of CaP coatings in the presence and absence of proteins was also investigated. It was observed from this study that as-sputtered CaP coatings were amorphous. The 400 degrees C heat-treated CaP coatings exhibited low crystallinity (1.9% +/- 0.4%), whereas the 600 degrees C heat-treated CaP coatings were highly crystalline (67.0% +/- 2.4%). The increase of Ca/P ratio, PO4/HPO4 ratio, and the number of PO4 peaks were observed to be consistent with the increase in heating temperature and the degrees of coating crystallinity. Phosphorus ions released from CaP coatings decreased with the increase of crystallinity of CaP coatings. In addition, immersion of CaP coatings in media containing proteins resulted in an increase in P ions released as compared with coatings immersed in media without proteins. It was concluded that the degree of CaP coating crystallinity can be controlled by varying the postdeposition heat-treatment temperature. It was also concluded that, aside from coating crystallinity, dissolution and reprecipitation of the coatings can be controlled by knowing the presence of proteins in the media and PO4/HPO4 ratio within the coatings.

Effect of biofluid environment on the dissolution and flexural strength of calcium phosphate bone cements.

This study investigates the dissolution of calcium (Ca2+), phosphorus (P5+), and the transverse strength of commercially available calcium phosphate (CaP) bone cements after immersion in fetal bovine serum and a tissue fluid substitute. It was observed that although a continual increase in Ca2+ and P5+ dissolution was detected in all three test media throughout the 21-day period, no statistical difference existed in the overall Ca2+ release after incubating the cements in the three different media. However, P5+ release after immersion in Tris solution (0.37 +/- 0.02 microgram/mm2) and fetal bovine serum solution (0.347 +/- 0.06 microgram/mm2) was significantly higher when compared with P5+ released (0.03 +/- 0.002 microgram/mm2) in tissue fluid substitute. In addition, no significant difference in transverse strength was observed for samples immersed in the three solutions during the 21-day period. However, the transverse strength for immersed CaP cement bars at 37 degrees C was statistically greater than non-immersed bars set aside at room temperature for the 21-day period (7.78 +/- 1.82 N and 3.19 +/- 0.93 N, respectively). It was concluded from this study that the transverse strength of the CaP bone cements was not significantly affected by the dissolution process but by the temperature at which the bone cement was exposed.

Effect of protein on the dissolution of HA coatings.

The dissolution behavior of hydroxyapatite (HA) in the presence and absence of protein needs to be investigated in order to fully understand the initial cellular response to HA surfaces. In this study, HA coatings were characterized using X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy (FTIR) prior to protein study. Fibronectin and albumin adsorption study were also performed. Calcium and phosphorus released in the presence and absence of albumin were measured. pH of the solution was measured daily. From the materials characterization, it was observed that the coatings exhibit a HA-type structure, with traces of sodium on the surface. FTIR indicated the possible presence of carbonates on the coatings. From the adsorption study, the amount of albumin adsorbed (0.052+/-0.005 microg/mm2) was statistically higher than the amount of fibronectin adsorbed on HA surfaces (0.035+/-0.002 microg/mm2). Flame atomic absorption indicated a significantly higher calcium ions released initially for HA coatings incubated with proteins as compared to coatings in the absence of proteins. However, after 7 days incubation, no significant difference in calcium ions release was observed between the HA coatings in the presence and absence of proteins. Phosphorus dissolution on HA coatings was not significantly affected by the presence of proteins. Thus, it was suggested from this study that the initial dissolution properties of calcium ions from HA coatings was dependent on the media.

Surface characterization of radio-frequency glow discharged and autoclaved titanium surfaces.

To characterize titanium surfaces treated with radio-frequency glow discharge (RFGD) after media exposure, surface chemical analyses were performed using x-ray photoelectron spectroscopy. Auger electron spectroscopy, and Fourier transform infrared-reflection absorption spectroscopy (FTIR-RAS). The RFGD treatments resulted in a cleaner surface as compared to as-sputtered or as-autoclaved titanium specimens. The oxide thickness of RFGD-treated titanium specimens was not statistically different from the as-autoclaved and as-sputter cleaned titanium specimens. Exposure to a phosphate-buffered saline solution revealed a greater deposition of calcium and phosphorous on the RFGD-treated surfaces. Auger electron spectroscopy depth profiles showed that calcium and phosphorous ions diffused into the titanium oxide layer. The calcium and phosphorous deposits were identified as amorphous calcium phosphate compounds using FTIR-RAS. These results suggest that RFGD treatments of titanium enhance calcium and/or phosphate affinity because of an increase in elemental interactions at the surface, thereby resulting in the formation of amorphous calcium phosphate compounds.

Effect of surface topography of titanium on surface chemistry and cellular response.

Surface topography plays a critical role in the interaction of dental implants with adjacent tissues. It has been hypothesized that an increase in surface roughness will result in an increase in calcium and phosphorus deposition after immersion in a simulated physiological solution and will increase protein production and calcium uptake by osteoblast-like cells. With the use of a profilometer, titanium samples ground with 600 grit silicon carbide paper were observed to have an average roughness (Ra) value of 0.28 +/- 0.03 micron, whereas titanium samples polished with 0.3 micron Al2O3 exhibited a Ra value of 0.11 +/- 0.01 micron. X-ray photoelectron spectroscopy analyses indicated the presence of calcium, phosphorus, sodium, and chlorine on both surface conditions after immersion in a protein-free physiologic solution. No significant difference in calcium and phosphorus concentrations were observed between the 600 grit or Al2O3 polished titanium samples after immersion in solution. The Ca/P ratio for both 600 grit and Al2O3 polished titanium was in the range of 0.8 to 1.1 after 12 days in solution. The percent protein retained by the rat bone marrow cell layer on both the Al2O3 polished and 600 grit titanium surfaces increased dramatically during the initial 3 days of the study. The 45Ca assays revealed no significant difference in cellular calcification on Al2O3 polished and 600 grit titanium surfaces. For both the Al2O3 polished and 600 grit surfaces, a sharp increase in 45Ca incorporation was observed after 9 days incubation.