FOURIER-TRANSFORM INFRARED MICROSCOPY OF CALCIFIED TURKEY LEG TENDON

Fourier transform infrared microscopy (FT-IRMS) was used to monitor sp atial variations in the quality and quantity of the mineral phase in c alcified turkey tendon. Spectral maps were generated by analysis of 50 mu m x 50 mu m areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and miner alized matrix begins, revealed marked changes in the spectrally determ ined mineral-to-matrix ratio, whereas regions deeper into the minerali zation front showed a relatively constant ratio. Since spectra of EDTA -demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtra ction. The broad, relatively featureless contour of the mineral nu(1), nu(3) phosphate region (900-1200 cm(-1)) showed only subtle changes a t different stages of mineralization. Second derivatives of these spec tra were calculated and compared with those of synthetic, poorly cryst alline hydroxyapatite (HA). The peak positions seen in second-derivati ve spectra of the mineral near the transitional region were within +/- 2 cm(-1) of the least mature synthetic HAs whereas spectra of the min eral deeper into the mineralization front were within +/- 2 cm(-1) of the most mature synthetic HAs. Spectra from cross- and longitudinal se ctions at equivalent positions in the tendon, and polarized FT-IRMS da ta were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and lo ngitudinal sections of the tendon showed no major differences in eithe r the nu(1), nu(3) phosphate region or the amide I, II, or III compone nts (1200-1800 cm(-1)). However, polarized FT-IR spectra revealed dram atic differences in both of these regions. Despite these differences, second-derivative analysis of the nu(1), nu(3) regions revealed no sig nificant changes in the positions of the underlying bands used to char acterize the environments of the phosphate ion in poorly crystalline H A. The results of this study demonstrate the power of FT-IRMS to monit or spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to t he collagen matrix.