An FT-IR microscopic investigation of the effects of tissue preservation on bone

Summary Fourier transform infrared microscopy is a powerful tool for the characterization of mineral and protein in histologic sections of bone. This study was concerned with determining whether techniques used to preserve these tissties and to prepare them for sectioning had an effect on spectral properties. The ₁, ₃ phosphate bands in the 900–1200 cm^-1 spectral region were used to evaluate the structure of the apatitic mineral in fresh-frozen, ethanol-fixed, and formalin-fixed 35-day-old rat femurs; fresh-frozen and formalin-fixed 20-day-old fetal rat femurs; ground 35-day-old rat diaphyseal bone samples; and formalin-fixed, methacrylate-embedded ground diaphyseal bone. The crystallinity (crystal size and perfection) of the bone apatite was assessed by a curve-fitting analysis of the ₁, ₃ phosphate bands. Results indicate that ethanol or formalin fixation of the 35-day-old intact rat femur, and formalin fixation and embedding of the ground rat bone do not significantly alter the crystallinity of the apatite. However, formalin fixation of the fetal rat bone did alter the structure of the apatite mineral phase. In addition, evaluation of protein secondary structure in the 35-day-old rat femur from the Amide I and Amide II vibrations near 1650 and 1550 cm^-1, respectively, revealed that protein conformation was altered by ethanol fixation.     

Fourier transform infrared microscopy of calcified turkey leg tendon

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 μm ×~ 50 μm areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization 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 subtraction. The broad, relatively featureless contour of the mineral ν₁,ν₃ phosphate region (900–1200 cm⁻¹) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within ±2 cm⁻¹ of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within ±2 cm⁻¹ of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the ν₁,ν₃ phosphate region or the amide I, II, or III components (1200–1800 cm⁻¹). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the ν₁,ν₃ regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor 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 the collagen matrix. Received: 17 January 1995 / Accepted: 26 May 1995     

Correlated physicochemical and age changes in embryonic bovine enamel

Summary Whole enamel scrapings from unerupted teeth of embryonic calves have been separated into fractions of varying density by stepwise centrifugation in bromoform-toluene mixtures of increasing specific gravity. Partition of enamel in this manner yields individual fractions of increasing mineral phase age and maturation. Whole scrapings and isolated fractions of the fetal bovine enamel were examined by X-ray powder diffraction, scanning electron microscopy, and atomic absorption and infrared spectroscopy to determine time-related changes in the physicochemical nature of the constituent mineral phase particles. These analyses showed poorly crystalline hydroxyapatite (HA) as the only detectable solid phase of calcium phosphate present in all fractions, its degree of crystallinity increasing with increasing density. Molar Ca/P ratios and magnesium content were highest in lowest density fractions. Carbonate vibration bands at 875 and 1420–1450 cm⁻¹, common to mineralized tissue, were observed in intermediate and higher density fractions and in whole unfractionated enamel. Another carbonate band at ∼705 cm⁻¹, unusual to vertebrate calcified tissue, was detected in low density fractions and disappeared rapidly with increasing enamel maturation. Its precise relation with the enamel mineral phase has not been determined.     

Distraction Osteogenesis Enhances Remodeling of Remote Bones of the Skeleton: A Pilot Study

Bone injuries have a systemic influence on the remodeling of bone. This effect has not been examined concerning its extent and duration. We measured the systemic effect of distraction osteogenesis on the remodeling of bones of the axial skeleton by means of the mineral apposition rate and bone formation rate in an animal experiment. Distraction osteogenesis was performed on the tibiae of 24 mature Yucatan minipigs. After a 4-day latency period, the tibiae were distracted 2 mm/day for 10 days. The ensuing consolidation phase lasted 10 days. Three fluorescent labeling substances were applied intravenously: calcein green at the second postoperative day, tetracycline 1 day after the end of the distraction phase, and xylene orange 2 days before sacrifice. We prepared ground sections from the ninth right ribs. The mineral apposition rate and bone formation rate were measured histomorphometrically on labeled osteons. The median mineral apposition rate during distraction was 2.39 μm/day (2.12–2.62 μm/day), which was higher than the rate during consolidation (median, 1.62 μm/day; 1.54–1.84 μm/day). The median bone formation rate confirmed this result and was 840.51 μm²/day (744.20–1148.26 μm²/day) during distraction and 384.25 μm²/day (330.84–467.71 μm²/day) during consolidation. Thus, a short period of distraction osteogenesis appears to have an anabolic effect on the mineral apposition rate of remote cortical bone.     

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 (PO₄ ³⁻) 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.     

Vibrations produced during erbium:yttrium–aluminum–garnet laser irradiation

The purpose of this study was to evaluate vibrations induced by an erbium:yttrium–aluminum–garnet (Er:YAG) laser in the non-contact mode and compare the vibrations with different pulse durations and energy parameters. The experiment was conducted on an extracted tooth built up in silicone impression material. The vibrations were measured by piezoelectric accelerometer for a super-short pulse (SSP), a very short pulse (VSP), and a short pulse (SP) at a frequency of 5 Hz for 1 s. For VSP and SP, the energy parameters tested were 200 mJ, 300 mJ, and 400 mJ. Measurements were performed 15 times for each individual irradiation energy level. The highest values of vibrations were measured for SP (0.160 ± 0.04 m/s²), and the lowest were measured for VSP mode at the energy parameter 200 mJ (0.05 ± 0.02 m/s²). There was a statistically significant (P < 0.01) difference between the various laser pulse modes (SSP, VSP, SP) at different energy parameters. At energy levels of 300 mJ and 400 mJ, the least amount of vibration during cavity preparations with the non-contact Er:YAG laser was produced by SSP mode.     

Influence of creatine supplementation on bone quality in the ovariectomized rat model: an FT-Raman spectroscopy study

The influence of creatine (Cr) supplementation on cortical and trabecular bone from ovariectomized rats was studied using FT-Raman spectroscopy. The intensity of organic-phase Raman bands was compared to mineral phase ones. Twenty-one female Wistar rats aged 3 months were divided into three groups (n = 7 per group): ovariectomized (OVX), ovariectomized treated with creatine (CRE) and sham-operated (SHAM) groups. Creatine supplementation (300 mg kg⁻¹ day⁻¹) was provided for 8 weeks, starting 12 weeks after ovariectomy. FT-Raman spectroscopy was performed on the right medial femoral mid-shaft (cortical bone) and third lumbar vertebral body (trabecular bone). The integrated intensities of mineral phase (phosphate and carbonate bands at 959 and 1,071 cm⁻¹, respectively) and organic phase (amide I band at 1,665 cm⁻¹) Raman bands were analyzed. The mineral-to-matrix (phosphate/amide I), carbonate-to-phosphate, and carbonate-to-amide I ratios were analyzed to assess bone quality. The phosphate content on trabecular bone was higher in the CRE group than the OVX group (p < 0.05). No significant changes in mineral or organic phases on cortical bone were observed. A radiographic assessment of bone density was encouraging as the same findings were showed by Raman intensity of phosphate from cortical (r ²  = 0.8037) and trabecular bones (r ² = 0.915). Severe ovariectomy-induced bone loss was confirmed by FT-Raman spectroscopy. The results suggest that the chemical composition of trabecular bone tissue may be positively influenced by Cr supplementation after ovariectomy.     

Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging

Summary The environment of CO₃ ²⁻ ions in the bone mineral of chickens of different ages and in bone fractions of different density have been investigated by resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopy. Three carbonate bands appear in thev ₂ CO₃ domain at 878, 871, and 866 cm⁻¹, which may be assigned to three different locations of the ion in the mineral: in monovalent anionic sites of the apatitic structure (878 cm⁻¹), in trivalent anionic sites (871 cm⁻¹), and in unstable location (866 cm⁻¹) probably in perturbed regions of the crystals. The distribution of the carbonate ions among these locations was estimated by comparing the intensities of the corresponding FTIR spectral bands. The intensity ratio of the 878 and 871 cm⁻¹ bands remains remarkably constant in whole bone as well as in the fractions obtained by density centrifugation. On the contrary, the intensity ratio of the 866 cm⁻¹ to the 871 cm⁻¹ band was found to vary directly and decreased with the age of the animal. In bone of the same age, the relative content of the unstable carbonate ion was found to be highest in the most abundant density centrifugation fraction. A resolution factor of the CO₃ ²⁻ band (CO₃ RF) was calculated from the FTIR spectra which was shown to be very sensitive to the degree of crystallinity of the mineral. The crystallinity was found to improve rapidly with the age of the animal. The CO₃ RF in the bone samples obtained by density centrifugation from bone of the same animal was found to be essentially constant. This indicates a fairly homogeneous, crystalline state of the mineral phase. A comparison of the maturation characteristics of synthetic carbonated apatites with bone mineral indicates that a simple, passive, physicochemical maturation process cannot explain the changes observed in the mineral phase of whole bone tissue or in the density centrifugation fractions of bone during aging and maturation.     

A resolution-enhanced Fourier Transform Infrared spectroscopic study of the environment of the CO3 2− ion in the mineral phase of enamel during its formation and maturation

Summary A resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopic study of the CO₃ ²⁻ ion in pig enamel of increasing age and maturity has demonstrated the existence of four different, main carbonate locations. The major CO₃ ²⁻ site arises as a result of the substitution of CO₃ ²⁻ ions in the positions occupied by PO₄ ³⁻ ions in the apatitic lattice. In addition, two minor locations have been identified in positions in which the CO₃ ²⁻ ions substitute for OH⁻ ions. The fourth carbonate group appears to be in an unstable location. Its concentration has been found to decrease with aging and maturation, during which there is a progressive increase in the amount of mineral deposited in the enamel. The distribution of the carbonate ions in the different apatitic sites varies randomly during the formation of the mineral phase in enamel and during its maturation. Although these changes have been shown to be related to changes in the composition of the mineral phase, a comparison of the parameters assessing the degree of crystallinity of the mineral phase from υ₂CO₃ ²⁻ and υ₄PO₃ ²⁻ infrared absorption data reveals a significant discrepancy related to the nonhomogeneous partition of the CO₃ ²⁻ ion in the mineral phase. After maximum mineralization is reached, the composition of the mature mineral phase is decidedly different than that of the initial mineral deposited; the changes affect principally the concentrations of Ca²⁺, OH⁻, and HPO₄ ²⁻ ions, but not the CO₃ ²⁻ ions.     

The association of pregnancy history with areal and volumetric bone mineral density in adolescence

Introduction Studies demonstrate that pregnancy may interfere with bone mineral density. Adolescence is a crucial time of life for bone mass acquisition and there are some questions as to the influence of pregnancy on bone mass at this age. Objective To evaluate the association between pregnancy history and areal (BMD) and volumetric (vBMD) bone mineral density in adolescence. Subjects and methods A cross-sectional study of 119 adolescents ranging from 12–20 years of age was conducted; 30 of these girls had a history of full-term pregnancy. The adolescents were selected during a routine visit to the Adolescent Gynecology Outpatient Facility, completed a questionnaire, and had a physical examination to evaluate weight, height and Tanner stage. Bone mineral densitometry of the lumbar spine (L₁–L₄) and total body (TB) was performed to measure bone mineral density and body composition. Results The mean measurements of the area, bone mineral content (BMC), BMD and vBMD of L₁–L₄ and the area, BMC and BMD of TB were not significantly different between adolescents with and without a pregnancy history, stratified by chronologic and gynecologic age. The percentage of adolescents with altered Z-scores was similar in both groups, and the prevalence ratio showed no association between pregnancy history and low bone mass (PR=0.52; CI 0.04–6.07). Upon multiple regression analysis, body mass index (BMI) and lean body mass (LBM) were the main factors associated with lumbar spine and total body measurements. Pregnancy history was inversely associated with areal BMD of L₁–L₄ (R ²=0.04) and vBMD of L₁–L₄ (R ²=0.04), accounting for only 4% variation in the lumbar spine. Conclusion These data suggest that adolescent pregnancy seems to exert no significant influence on the acquisition of bone mass and does not appear to represent a risk factor for osteoporosis in the future.