Microstructural Changes of Bone after Soft Tissue Removal: ATR-FTIR and XRD Spectroscopies

. Te structural alterations that may arise in boiled and nonboiled bones are often overlooked despite their critical importance in the development of defeshing techniques across various scientifc disciplines. To elucidate the microstructural characteristics of bones following the removal of soft tissue through conventional methods, attenuated total refectance Fourier-transform infrared (ATR-FTIR) and X-ray difraction (XRD) spectroscopies were employed. Our fndings indicate that the boiled water method resulted in higher crystallinity, as evidenced by the FTIR data, whereas the XRD data revealed the opposite. Tis underscores the notion that a direct comparison between these two techniques is unfeasible as they measure distinct crystallinity parameters. In addition, the cold water maceration method caused a signifcant reduction in collagen crosslinking, as evidenced by the lower index observed.

FTIR is a powerful technique that enables the analysis of bone composition at the molecular level [11][12][13][14].It measures the absorption and transmission of infrared radiation by bone samples, providing information about the functional groups, organic molecules, and mineral content present in bones.FTIR can identify the presence of collagen, which is the predominant organic component of bones, as well as other biomolecules, such as lipids, proteins, and carbohydrates [15,16].It can also provide insights into the chemical bonds, crystalline structure, and mineralization process of bones, making it a valuable tool for assessing bone quality, health, and disease [17].
On the other hand, XRD is a technique that analyzes the crystalline structure of bone samples.It measures the diffraction of X-rays by the crystal lattice of bone minerals, such as hydroxyapatite, which is the main inorganic component of bones [18][19][20][21].XRD provides information about the crystallographic orientation, size, and arrangement of mineral crystals in bones, which is critical in understanding their mechanical strength, stifness, and mineralization patterns [22].XRD is highly sensitive to changes in bone mineral crystallinity, which can refect alterations in bone development, aging, and pathology [23,24].
Despite their diferences, FTIR and XRD are highly complementary in bone studies.FTIR provides information about the organic and mineral content of bones, while XRD focuses on the crystalline structure of bone minerals.Together, they provide a comprehensive understanding of bone composition and structure, enabling researchers to obtain a holistic view of bone properties.For example, FTIR can reveal changes in the collagen content and structure, while XRD can provide insights into alterations in mineral crystallinity.Tis combined approach allows for a more comprehensive analysis of bone health, disease, and evolution.
However, it is important to note that FTIR and XRD can sometimes yield opposing results in terms of bone crystallinity.FTIR measures the absorption and transmission of infrared radiation, which can be afected by various factors, including the organic components of bones, water content, and sample preparation [25,26].As a result, FTIR can provide an overestimation or underestimation of bone crystallinity, depending on the specifc conditions of the analysis.On the other hand, XRD directly measures the difraction of X-rays by the crystal lattice of bone minerals, which is not infuenced by the organic components of bones or water content [18].Terefore, XRD is considered a more accurate technique for assessing bone crystallinity.
To reconcile the diferences in bone crystallinity results obtained from FTIR and XRD, researchers often use calibration techniques, such as the conversion of FTIR spectra to a quantitative crystallinity index (CI) based on XRD data [27].Tis allows for more accurate comparison and interpretation of results obtained from both techniques.In addition, other techniques, such as Raman spectroscopy and solid-state nuclear magnetic resonance (NMR), can also be used in conjunction with FTIR and XRD to further validate and corroborate fndings related to bone studies [27,28].
Hence, the primary objective of this study is to comprehensively explore the critical role of FTIR XRD in advancing bone research and elucidate the complementarity of these techniques in the bone study.Furthermore, this study also aims to investigate potential disparities in bone crystallinity outcomes that may arise when utilizing FTIR and XRD methods to provide a more comprehensive understanding of bone mineralization processes.By conducting a detailed analysis of these techniques, their strengths, limitations, and synergistic efects in bone research, this study seeks to contribute to the advancement of knowledge in the feld of bone science and provide valuable insights for future research endeavors.

Sample Preparation.
A complete femur bone from a single camel was purchased from a local retailer located in Irbid, Jordan.Te femur bone was sectioned into cylindrical shapes and underwent tissue removal using a scalpel.Te cylindrical bone pieces were divided into two groups, each consisting of ten randomly selected samples, for comparison of the microstructure.Te frst group underwent treatment using the bacterial cold water maceration technique, wherein the samples were immersed in a closed container flled with distilled water at room temperature for one week, with the water being replaced every two days to prevent mold growth [29].Following cleaning with distilled water, the bone samples were air-dried for 48 hours at room temperature.
Te second group, comprising 10 samples, underwent treatment via the bacterial warm water maceration technique.Te bone samples were placed in a closed container flled with distilled water, which was brought to a boil, and were subjected to this temperature for 3 hours [30].Following this, the samples were left in the container for 24 hours, allowing them to cool to room temperature.Te boiled bone samples were then washed with distilled water and air-dried for 48 hours at room temperature to ensure consistency across all samples.Subsequently, all samples from both groups underwent analysis using ATR-FTIR spectroscopy and X-ray difraction.

ATR-FTIR Spectroscopy.
An FTIR instrument (TEN-SOR II, platinum ATR, Bruker Optik GmbH) equipped with a mercury-cadmium-telluride detector (MCT) was employed to obtain spectral data.Te spectral range between 400 and 4000 cm −1 was scanned, as it corresponds to organic and inorganic compounds in bone.Each sample spectrum was collected as an average of 24 scans at a resolution of 4 cm −1 .To identify peak positions in our proposed spectral data, we used OriginPro software (version 2020) and applied a modifed Savitzky-Golay flter with a window size of 11 and a polynomial order of 2 to the raw data.Tis flter was used to overcome background, dark current noises, and baseline drift.Te second derivative method was applied to locate peak maxima and minima, and we used a threshold of 0.1 and a prominence criterion of 0.05 to flter out false peaks.
To determine the number of peaks to ft, we frst examined the expected spectral features of our samples, which included the amide I band and the phosphate region.We compared our spectra to relevant literature references and previous experimental data to identify potential peak positions and assignments.We also performed a statistical analysis using a Gaussian mixture model to estimate the number of underlying peaks in the data.Based on these analyses, we selected ten peaks to ft in the amide I region and eleven peaks in the phosphate region.Te band position centers were chosen to capture the major spectral features of our samples and were consistent with previous studies of similar compounds.We note that our peak identifcation and ftting approach may be infuenced by the specifc software and parameters used and may be subject to some degree of uncertainty.

X-Ray Difraction (XRD). Te X-ray difraction (XRD)
patterns of macerated bone were obtained using a Shimadzu LabX, XRD-6000 X-ray difractometer in the 2θ range of 10-70 °(Bragg angle) with a step size of 0.02 °/min and a scan speed of 3.0 deg./min.Te XRD measurements were 2 Journal of Spectroscopy performed using a 40 kV and 20 mA X-ray difractometer that generated CuKα radiation with a wavelength (λ) of 1.5406 °A.Te raw data were analyzed using Origin software.

Statistical Analysis.
Te crystallinity of the bone was determined through the 1020/1030 cm −1 ratio and full width at half maximum (FWHM) at 604 cm −1 , while collagen crosslinking (1660/1690 cm −1 ) was analyzed by infrared spectroscopy.XRD was used to measure crystallinity and crystal size at the 002 patterns.Statistical analysis was performed to compare the results obtained from cold water and boiled water bone maceration methods for each analysis, using a two-sample t-test.

ATR-FTIR and XRD Spectra.
Te ATR-FTIR spectra and the band positions of the relevant spectral features are shown in Figure 1.IR spectra were carried out in the frequency range of 400 to 4000 cm −1 to establish potential diferences between the two bone maceration methods used in this study.It is important to note that none of the treatments resulted in the disappearance or appearance of new IR bands.Only diferences in IR band intensity were observed.
Tere were strong bands at 558 and 604 cm −1 , which were attributed to PO4 3− υ4.Another prominent band was at 1012 cm −1 , which was assigned to HPO4 2− .Vibrational bands corresponding to the amide group and CO3 2− were also evident.In the bone, there are peaks related to amides and organic matrix (CH bands).
To deconvolute overlapping specifc bands, the deconvolution analysis technique (DAT) is utilized using Ori-ginPro software (version 2020), as demonstrated in Figure 2. Te position of bands was determined through previously published and analyzed similar materials from the literature and by calculating second derivatives.Gaussian band shapes and linear baselines were employed as input parameters on normalized spectral data.Te number of bands was taken as minimum as possible to reach a regression coefcient r 2 value near unity [32][33][34][35][36].
Figure 3 displays the XRD spectra of the macerated bone.Te crystalline phases present in the samples were identifed by XRD using standard JCPDS 9-0432 cards available in the system software.Te XRD spectra of the bone samples exhibited several peaks with strong intensities at angles between 25 ≤ 2θ ≤ 26, which correspond to the crystal plane 002, and angles between 30 ≤ 2θ ≤ 34, which correspond to the crystal plane 211.However, the profles exhibited similar peak positions with diferences in peak intensity and width.In this study, we focused on the 002 pattern in our calculations to compare the crystallinity and calculate the crystal size of the bone samples after the maceration process.

IR Analysis of Crystallinity Index and Collagen Crosslinks in Bones.
Bone crystallinity was determined using the traditional band intensity ratio of 1030/1020 cm −1 and the full width at half maximum (FWHM) of the peak at 604 cm −1 .Te collagen crosslinking index was estimated by the intensity ratio of the subbands at 1660 and 1690 cm −1 .Tese parameters were statistically analyzed and compared between the two methods (cold water and hot boiled water bone maceration) using a two-sample t-test for each analysis.Te mean values of the crystallinity and collagen crosslinking in the bone samples are presented in Table 1.
In the IR analysis, the mean values of the crystallinity based on the 1030/1020 cm −1 band intensity ratio for the boiled water group were slightly higher than those of the cold water group, although the diference was relatively small, it was statistically signifcant (p � 0.008), as determined by the t-test.Te mineral crystallinity index calculated based on the FWHM of the peak at 604 cm −1 is inversely proportional to the bandwidth of the peak.Tis peak provides direct information on the crystallinity index of the sample and corresponds to the apatitic phosphate environment.Te narrower the peak, the higher the crystallinity index.Our analysis revealed that the mean values of the crystallinity based on the FWHM of the 604 cm −1 peak and the collagen crosslinking were signifcantly lower in the boiled water group than in the cold water group (p < 0.001).

XRD Analysis of Crystallinity and Crystal Size.
Crystallinity is directly related to the degree of perfection and size of crystals, which is refected by the inverse width of the (002) (c-axis refection) in the bone mineral powder Xray difraction pattern.Te Debye-Scherrer approximation [37] is used to calculate the average crystal size (D) from XRD data.Bragg's constant (k � 0.94), the full width at half maximum of the peak in radians (β), the X-ray wavelength (λ � 0.1540598 nm for CuKα), and the 2θ difraction angle of the 002 peak (θ) are the parameters involved in the calculation: In the XRD analysis, as presented in Table 2, the mean value of the crystallinity at FWHM 002 was found to be higher in the boiled water group than in the cold water group, but the crystal size was found to be smaller in the boiled water group than in the cold water group.Tese diferences were statistically signifcant (p � 0.001 and p � 0.002, respectively).Te individual sample readings within each group were observed to be very close to each other, resulting in a relatively small standard deviation in each group of the study.

Discussion
In this study, we assessed the variations and disparities in the organic and inorganic bone structure concerning the defeshing process, which was based on boiled water and cold water maceration.Two diferent analytical techniques were employed.Te FTIR results indicated a signifcant change in bone crystallinity between the two maceration methods that were utilized to eliminate the soft tissue from the bone.Te intensity ratio of 1030/1020 cm −1 was found to be higher in the boiled bone, and simultaneously, the Journal of Spectroscopy FWHM of the peak at 604 cm −1 was lower in the boiled bone.Tese fndings suggest that the crystallinity of the boiled bone samples is greater than that of the bone samples that were treated with the cold water maceration method.
Previous research consistently demonstrates that hydroxyapatite changes to a purer form with larger crystals, higher crystallinity, and reduced porosity as the burning temperature increases [38], owing to the release of carbonate    4 Journal of Spectroscopy and water content [39].In addition to the change in crystal size, there is also a signifcant alteration in the shape of the crystal, which occurs primarily between 500 °C and 700 °C [40,41].Although most studies on heated bone have focused on burned bone, only a few studies have been conducted on boiled bone.Consequently, there is a diference in the response of the bone when it is boiled as opposed to burning.Terefore, most studies on boiled bone have examined the change in the organic component of the bone, and there is evidence to suggest that there is a diference between the bone boiled in seawater and freshwater [42].Te fndings suggest that the crystallinity of bone samples subjected to boiling was higher than those treated by the cold water maceration method, as per the results obtained through FTIR spectroscopy.It is important to note that these results are linked to the crystal bonds of the samples [27].However, the XRD data revealed the opposite trend.Te crystallinity index of the boiled bone samples was lower than that of the cold bone samples.Tese results are associated with the crystal structure arrangement of the samples [27].In addition, the XRD data suggested a larger crystal size of bone samples in the cold water maceration method.Despite the increased use of crystallinity measures in bone structure studies, it remains uncertain whether these methods can be efectively applied without considering all the issues associated with their use and application.In other words, although various techniques can provide crystallinity parameters, the XRD values cannot be directly compared to those obtained from FTIR or Raman spectroscopy [43][44][45].
Crosslink chemistry is a crucial characteristic of type I collagen in the bone, providing it with high tensile strength and viscoelasticity through intermolecular crosslinking.FTIR has been employed to obtain insights into the protein structure through the underlying bands of the amid spectral   region.Of particular interest is the ratio of the sub-bands (1660/1690 cm −1 ), which serves as a measure of the collagen crosslinks in the bone structure.Te 1660 cm −1 band is associated with the presence of α-helix nonreducible (mature) collagen crosslinks, while the 1690 cm −1 band is linked to the reducible (immature) crosslinks.Table 1 demonstrates that the collagen crosslinking index of macerated bone samples subjected to the boiled water method was lower than that of bone samples macerated in cold water.Tis indicates that temperature is a crucial factor in determining the organic bone profle.Specifcally, the reduction in the crosslinking index is due to the decrease in the degree of molecular order of collagen [46].Terefore, understanding the extent of collagen crosslinking in the bone is crucial in comprehending the overall impact of microstructural diferences in the bone.
Te fndings of this study are limited due to several factors.Firstly, the use of specifc animal remains as samples could afect the generalizability of the results.Although it has been previously suggested that a basic animal model of the bone can be employed to study the human bone due to the similarity in chemical composition between humans and mammals, there exist some diferences in the microstructure of human and camel bones that could have an impact on the outcomes of this research.Secondly, while the study provides insight into the performance of two defeshing techniques and their efects on the bone tissue, conducting additional defeshing techniques could confrm the results.Lastly, further assessment of bone alteration techniques such as scanning electron microscopy (SEM) and Raman spectroscopy could be carried out to ofer more comprehensive knowledge on the changes that could occur in the bone microstructure.

Conclusion
Tis study seeks to contribute to the understanding of the importance of using FTIR and XRD in bone research, their complementarity, and how potential discrepancies in bone crystallinity results can be reconciled.Te fndings of this study may have implications in various scientifc felds, including biomedical research, paleontology, archaeology, and forensic science, enhancing our understanding of bone properties, health, disease, and evolution.
According to the FTIR data, bones treated with the boiled water method exhibited greater crystallinity, but the XRD data indicated the opposite.Tis suggests that a direct comparison between these two techniques is not feasible, as they measure diferent parameters of crystallinity.In addition, the collagen crosslinking was noticeably altered, with a reduced index observed in the cold water maceration method.

Figure 1 :
Figure1: Te ATR-FTIR spectra of bone maceration with boiled water (red) and bone maceration with cold water (black).Te main infrared bands are assigned[31].

Figure 2 :
Figure 2: Curve-ftting analysis of the phosphate band (a) and amide I profle (b) of the IR bone spectra illustrated the subbands.Te experimental spectrum (black) is overlaid with the reconstructed envelope (red), which is composed of ftted curves (green).

Figure 3 :
Figure 3: Te XRD spectra of bone samples subjected to maceration with boiled water (red) and cold water (black).

Table 1 :
IR analysis of bone crystallinity and collagen crosslinking parameters (mean ± SD).

Table 2 :
XRD analysis of bone crystallinity and its crystal size at 002 pattern (mean ± SD).