F. Betts

FTIR microspectroscopic analysis of human osteonal bone

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of non-osteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 μm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate v1,v3 band at 900–1200 cm-1 to the amide I band at 1585–1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the v2 band at ≈850–900 cm-1) to phosphate v1,v3 ratio decreased as the mineral to matrix ratio increased. Analysis of the v2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in “labile” carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the v1,v3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at ≈1020 and ≈1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.

Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: New correlations between X-ray diffraction and infrared data

Fourier Transform infrared spectroscopic analysis of maturing, poorly crystalline hydroxyapatite (HA) formed from the conversion of amorphous calcium phosphate (ACP) at constant pH or variable pH show only subtle changes in the ν1, ν3 phosphate absorption region (900 cm−1−1200 cm−1). This region is of interest because it can ve detected by analysis of mineralized tissue sections using FT-IR microscopy. To evaluate the subtle spectral changes occurring during the maturation, second derivatives of the spectra were calculated. HA formed at constant pH showed little or no variation in the second derivative peak positions with bands occurring at 960 cm−1, 985 cm−1, 1030 cm−1, 1055 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1. These bands can be assigned to molecular vibrations of the phosphate (PO43−) moiety in an apatitic/stoichiometric environment of HA. In contrast, during the early stages of maturation of the HA formed at variable pH, second derivative peak positions occurring at 958 cm−1, 985 cm−1, 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 shifted in position with maturation, indicating, that the environment of the phosphate species is changing as the crystals mature. Peaks at 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 were attributable to nonstoichiometry and/or the presence of acid phosphate-containing species. This concept was supported by the lower Ca:P molar ratios measured by chemical analysis of the synthetic material made at variable pH. Using the second derivative peak positions as initial input parameters, the ν1, ν3 phosphate region of the synthetic HAs prepared at constant pH were curve fit. X-ray diffraction patterns of these same materials were also curve fit to calculate the changes in crystallinty (size/perfection) in the c-axis 002 reflection as well as the 102, 210, 211, 112, 300, 200, and 301 planes. Linear regression analysis showed that the changes in the percent area of the underlying bands at 982 cm−1, 999 cm−1, 1030 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1 were correlated with changes in crystallinity in one or more of the reflection planes. It is suggested that a combination of second-derivative and curve-fitting analysis of the ν1, ν3 phosphate contour allows the most reproducible evaluation of these spectra.

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the ν4 PO43- vibration

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.

Post-irradiation aging of ultra-high molecular weight polyethylene.

A study was performed to determine the time-course of oxidative degradation and the extent to which the degradation proceeded through the bulk of ultra-high molecular weight polyethylene joint components that had been irradiated and stored on a shelf. Standardized cylindrical samples, taken from a single batch of extruded polyethylene, were cleaned, packaged, and sterilized according to protocols used for commercial joint-replacement components. After sterilization, the samples were stored in the packages for time-periods of one day to more than one year. At each interval studied, thin sections were cut as a function of depth into the bulk of the sample and were used to determine the density and the infrared spectra. Marked alterations in the density and the infrared spectra consistent with continuing oxidative degradation occurred throughout the year of storage on the shelf. The alterations were most severe near the surface of the samples.

Polyethylene and metal debris generated by non-articulating surfaces of modular acetabular components

We report a prospective study of the liner-metal interfaces of modular uncemented acetabular components as sources of debris. We collected the pseudomembrane from the screw-cup junction and the empty screw holes of the metal backing of 19 acetabula after an average implantation of 22 months. Associated osteolytic lesions were separately collected in two cases. The back surfaces of the liners and the screws were examined for damage, and some liners were scanned by electron microscopy. The tissues were studied histologically and by atomic absorption spectrophotometry to measure titanium content. The pseudomembrane from the screw-cup junction contained polyethylene debris in seven specimens and metal debris in ten. The material from empty screw holes was necrotic tissue or dense fibroconnective tissue with a proliferative histiocytic infiltrate and foreign-body giant-cell reaction. It contained polyethylene debris in 14 cases and metal in five. The two acetabular osteolytic lesions also showed a foreign-body giant-cell reaction to particulate debris. The average titanium levels in pseudomembranes from the screw-cup junction and the empty screw holes were 959 micrograms/g (48 to 11,900) and 74 micrograms/g (0.72 to 331) respectively. The tissue from the two lytic lesions showed average titanium levels of 139 and 147 micrograms/g respectively. The back surfaces of the PE liners showed surface deformation, burnishing, and embedded metal debris. All 30 retrieved screws demonstrated fretting at the base of the head and on the proximal shaft. Non-articular modular junctions create new interfaces for the generation of particulate debris, which may cause granulomatous reaction.

Stabilization of Amorphous Calcium Phosphate by Mg and ATP

SummaryA synergistic effect has been demonstrated when magnesium and adenosine triphosphate (ATP) are used together in solution to delay the conversion of a slurry of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA). Conversion is delayed in some instances more than 10 times as long as with either ATP or Mg alone. In all experiments conversion did not begin until ATP in solution had decreased through hydrolysis to an undetectable level. The effect of Mg is to decrease substantially the rate at which ATP hydrolysis occurs. Once conversion began it proceeded more slowly in the presence of both Mg and ATP than with Mg or ATP alone. ATP was also found to prevent the formation of HA from metastable solutions of calcium and phosphate which did not contain any solid phase. Over the time period of these experiments, ATP hydrolyzed to a negligible extent in Tris-HCl buffer and in solutions containing Ca, PO4, and Ca plus PO4 ions. Hydrolysis of ATP does occur in the presence of ACP or HA, presumably by transphosphorylation on the surface of the solid calcium phosphate phase. It was concluded that ATP stabilized ACP, not by affecting its dissolution, but either by poisoning heteronuclear growth sites, or by poisoning the growth of embryonic HA nuclei (formed heterogeneously or homogeneously) before their critical size is reached, or by poisoning both. In the case of embryonic HA nuclei, the poisoned nuclei would go back into solution preventing HA crystal formation. In addition, it was found that the neutral Ca9(PO4)6 clusters, which are believed to be the basic structural unit of ACP, break down into individual Ca and PO4 ions when ACP dissolves in aqueous medium.

Effect of carbonate and biological macromolecules on formation and properties of hydroxyapatite

Amorphous calcium phosphate (ACP) was transformed at 25° to hydroxyapatite (HA) in horse and bovine serum; solutions of serum-protein fractions in tris-HCl buffer (pH 7.4), and pH 7.4 buffers containing from 0.1 to 10 times physiological CO32− concentration. The ACP-to-HA transformation was slower in whole serum and serum fractions than in control buffer solution. The observed adsorption of serum proteins on ACP and HA probably inhibits both the dissolution of the ACP particles and the growth of HA crystals. After 72 h all transformations were complete as determined by X-ray diffraction. The HA crystal dimensions decreased with increasing CO32− but the shape, as shown by X-ray linewidths, was relatively constant up to about 4% CO32−. At 15% CO32− the crystals were more equiaxial and less needle-like in habit. The radial distribution function (RDF) of HA with 3.7% CO32− is less well resolved than the RDF of HA with ambient CO32− (1.1%). The peaks are less sharp and their amplitude falls more rapidly with increasing atomic separation than for low CO32−-HA. These effects show that CO32− decreases the regularity of the atomic arrangement when incorporated in HA. The rapid decrease, with increasing CO32− content, of the IR splitting of the P−O bending mode of CO32−-HA is attributed to reduced crystal size and possibly to a perturbation of the crystal field due to CO32−-induced lattice distortion. Finally, for bone mineral, it is probable that the poor resolution of the X-ray and IR patterns is due, in large part, to small crystal size and internal disorder caused by CO32−.

Size of metallic and polyethylene debris particles in failed cemented total hip replacements

Reports of differing failure rates of total hip prostheses made of various metals prompted us to measure the size of metallic and polyethylene particulate debris around failed cemented arthroplasties. We used an isolation method, in which metallic debris was extracted from the tissues, and a non-isolation method of routine preparation for light and electron microscopy. Specimens were taken from 30 cases in which the femoral component was of titanium alloy (10), cobalt-chrome alloy (10), or stainless steel (10). The mean size of metallic particles with the isolation method was 0.8 to 1.0 microns by 1.5 to 1.8 microns. The non-isolation method gave a significantly smaller mean size of 0.3 to 0.4 microns by 0.6 to 0.7 microns. For each technique the particle sizes of the three metals were similar. The mean size of polyethylene particles was 2 to 4 microns by 8 to 13 microns. They were larger in tissue retrieved from failed titanium-alloy implants than from cobalt-chrome and stainless-steel implants. Our results suggest that factors other than the size of the metal particles, such as the constituents of the alloy, and the amount and speed of generation of debris, may be more important in the failure of hip replacements.

Particulate metallic debris in cemented total hip arthroplasty.

Several studies conducted by the authors in the last six years demonstrate that the generation of metallic debris is more severe with titanium alloy than with cobalt-chrome alloy femoral components in cemented total hip arthroplasty (THA). The debris is generated from the articulating surface, particularly if entrapped acrylic debris produces three-body wear, and from the stem surface when the component loosens and abrades against fragmented cement. In selected cases in which the titanium metallic debris is copious, premature failure and severe progressive bone loss occurs. Electron microscopy demonstrates that the particles of metallic debris can be extremely small (a few hundredths of 1 micron). They are phagocytized by the macrophages and transported to the phagolysosomes. In this highly corrosive environment, the very high surface area of the particles may release toxic concentrations of the constituents of the alloy intracellularly, probably leading to progressive cell degeneration and death, with subsequent release of intracellular enzymes and ingested metallic debris. This cycle most likely repeats itself, leading to tissue necrosis. The results presented do not support the use of titanium alloy femoral components for cemented THA, particularly for the articulating surface.

Amorphous calcium phosphate in casein micelles of bovine milk

SummaryThe calcium phosphate remaining after hydrazine deproteination of casein micelles isolated from bulk skim milk exhibits under the electron microscope a very fine and uniform granularity being formed by small subunits with a true diameter of approximately 2.5 nm. This material, which is about 10 percent by weight citrate, termed calcium phosphate citrate (CPC) complex, also contains Mg and Zn at molar ratios of 0.03 and 0.003 respectively. Radial distribution function (RDF) and infrared analyses show that CPC is a Mg-containing amorphous calcium phosphate (ACP) similar to synthetic and cytoplasmic ACP. Presence of CPC in casein micelles as an amorphous colloid bonded with phosphoproteins provides the means for storing in milk large amounts of Ca (16 mM) and Pi (10 mM) in a readily utilizable form but at a higher ion concentration than found in biological solutions.