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2.5 Bone examination methods

Multiple factors contribute to bone quality, including geometric and material factors. Geometric factors include the macroscopy of the whole bone and the microscopic construction of the trabeculae. Material factors include bone composition and the arrangement of the primary

microstructural constituents as well as microdamage (27). Traditional radiography and dual-energy X-ray absorptiometry (DEXA) are cost-effective and widely available methods to assess geometry and structure.

Radiography can assess cortical thickness, which is a reliable indicator of bone quality and shows a positive correlation with bone mineral density (BMD) evaluated by DEXA (44). Three-dimensional (3D) microstructure level assessment is performed with the development of advanced imaging techniques. Various techniques are now available to evaluate bone

structure and mechanical properties, at multiple levels from the macroscopic to the microscopic and nanoscopic levels (Table 4).

Table 4. Common techniques for the assessment of bone quality at different levels (45,46).

Bone assessment methods Bone mechanical

properties – Mechanical methods

(Macro to Micro)

Bone geometry and microarchitecture – Imaging methods

(Macro to Micro)

Bone tissue composition – Imaging, Physical, and

Chemical methods (Micro to Nano) - Whole bone

mechanical testing - Bulk tissue specimen

testing - Microbeam testing - Microindentations - Nanoindentations

- Radiography - CBCT - DEXA - QCT

- High-resolution MRI - Micro-CT

- NMR imaging - FTIR

- Raman Spectroscopy - Atomic absorption spectroscopy

- Electron microscopy - Gravimetric analysis - Chemical analysis CBCT = Cone beam computed tomography, DEXA = Dual-energy X-ray

absorptiometry, QCT = Quantitative computed tomography, MRI - magnetic resonance imaging, NMR = Nuclear magnetic resonance, FTIR = Fourier Transform Infrared Imaging

2.5.1Micro-computed tomography (micro-CT)

Micro-CT is the gold standard method in bone quality assessment, as it allows the evaluation of bone microarchitecture with high resolution. The application of micro-CT imaging to the study of trabecular and cortical bone morphology in animal and human specimens has increased

tremendously in recent years. It has been suggested that the combination of micro-CT images with mechanical tests can provide quantitative and bone strength information on human bone specimens (47,48). The study specimen is rotated in angular increments between the x-ray source and detector in the micro-CT scanners. The obtained data at each position are reconstructed into a 3D array, which can be converted to mineral density values with the inclusion of appropriate calibration phantoms (49). The development of in vivo micro-CT scanners has allowed studying the macro- and micro-architecture of the bones of smaller living animals such as

rodents. High-resolution Synchrotron micro-CT with a spatial resolution of approximately 1 µm is mostly applied for the assessment of tissue mineral density (TMD), resorption spaces, and microcracks (46). Micro-CT is a non-invasive technique and can be used to measure some important

microstructural parameters, including bone volume fraction (Bone volume/Total volume, BV/TV), bone surface density (Bone surface/Total volume, BS/TV), specific bone surface (Bone surface/Bone volume, BS/BV), cortical bone area, cortical thickness, trabecular thickness, trabecular number, trabecular separation, porosity and connectivity density. Bone volume and bone surface in the above parameters are usually calculated for the segmented bone, whereas the total volume is calculated for the entire bone region of interest (50).

2.5.2Raman spectroscopy

The Raman effect underlying Raman spectroscopy was discovered in 1928 by Nobel Laureate Dr. CV Raman in India. (51) In the late 1970s, a

microscopic Raman with a laser optical microscope equipped with a Raman spectrometer was developed, which was used in many fields as an

analytical tool. With advancements in digital cameras, the performance of detectors improved, and with the development of spectrometers,

sensitivity has also been improved. It has become an analytical method for researchers in many fields.

Raman spectroscopy enables the assessment of the chemical

composition and tissue constituents of bone. In Raman spectroscopy, the incident light is focused on the specimen, which excites vibrations by the interaction of laser photons and the sample. This causes the scattering of light at a new, lower frequency which is determined by the energy of its characteristic molecular vibration peaks on the Raman spectrum, and it can be analyzed further for tissue composition (52). The use of Raman spectroscopy with an optical microscope and a focal plane array detector enables spatial mapping of microscale bone tissue composition (53).

The general Raman spectral band position of a bone sample is shown in Table 5. It presents the major mineral and matrix bands. At ~960 cm-1 the

phosphate ν1 band and at ~1070 cm-1 the B-type carbonate band are the important Raman mineral bands. The phosphate and carbonate bands provide information on crystal structure and crystallite size (54). Changes in these bands can result from mechanical deformation, genetic defects, or disease. Other important Raman matrix bands are the amide I at ~1660-1690 cm-1, the Proline - hydroxyproline bands at 853 cm-1 and 872 cm-1, the amide III at 1242-1272 cm-1, and the CH2 wag at 1446 cm-1. These represent the collagen matrix bands and are sensitive to changes in the protein structure or collagen hydrogen bonding. The Raman parameters to study the composition of bone are mineral-to-matrix ratio (MMR), mineral crystallinity, carbonate substitution and the collagen cross-linking ratio.

The MMR, measure of bone mineralization is usually calculated as the ratio of band height or area of the mineral contents such as phosphate or carbonate to that of the matrix contents such as amide or Proline

-hydroxyproline or Phenylalanine. Mineral crystallinity is calculated from the inverse of full width at half-maximum (FWHM) of the phosphate band and is an indicator of mineral crystal size. The carbonate substitution ratio is calculated as the intensity ratio of the carbonate to phosphate peaks (CPR), and it represents the abnormal type B carbonation. Collagen crosslinking ratio is the area or height ratio of amide I, 1690 cm-1 component to the 1660 cm-1 component (55,56) representing the ratio of non-reducible mature to reducible immature collagen.

Table 5. Raman spectroscopic band assignments for bone mineral and matrix components (57,58)

Raman shift, cm-1

Assignment Comments

430 ν2PO43- Strong band

450 ν2PO43- The shoulder on 430 cm-1 band 584 -590 ν4PO43- Multiple partially resolved components

609 ν4PO43- The shoulder on the 590 cm-1 band 853 ν(C-C) Collagen proline may include δ(C-C-H)

contribution from tyrosine 872 ν(C-C) Mostly collagen hydroxyproline 955 ν1PO43- Transient bone mineral (P-O) phase, usually

seen in immature bone

957 ν1PO43- Bone mineral containing extensive HPO42−, usually immature

959-962 ν1PO43- Bone mineral, mature

1003 ν(C-C) Phenylalanine

1035 ν3PO43- Overlaps with proline ν(C-C) component 1070 ν1CO32- Overlaps with the component of ν3PO4

3-1076 ν3PO43- Overlaps with a component of ν1CO3

2-1242 Amide III Protein β-sheet and random coils

1272 Amide III Protein α-helix

1293-1305 δ(=CH) Lipid band, sometimes seen in fresh untreated bone

1340 Amide III Protein α-helix sometimes called CH2CH2 wag 1446 δ(CH2) Protein CH2 deformation

1660 Amide I Strongest amide I ν(C=O) component, polarization-sensitive, mature cross-links 1690 Amide I Shoulder, prominent with immature cross-links.

The band also relates to β-sheet or disordered secondary structure

2.5.3Scanning electron microscopy (SEM)

SEM allows the study of bone structure at the microscale tissue level.

Conventional SEM operates at high vacuum, under clean and dry

conditions with electrically conductive samples. In the SEM technique, an electron beam is focused on the specimen surface and interacts with the atoms in the sample to generate signals. It can generate three types of

signals: secondary electrons (SEs), backscattered electrons (BSEs) and x-rays. The SE signals are generated when an incident electron imparts some of its energy to an atom present in the specimen. It results in the emission of a low-energy ionized electron from the specimen. This signal arising from the specimen surface provides the topographic detail (59). BSE signals are generated when an incident electron hits an atom in the specimen.

This causes a backward scattering and the obtained gray-level intensity values are related to the tissue microstructure (60). The quantitative BSE imaging (qBEI) technique is suitable to assess the bone mineral density distribution which reflects the bone turnover and mineralization. (61). The x-ray analysis includes Energy-dispersive x-ray microanalysis. In this

technique, when the incident electron beam hits the specimen, x-rays with energy characteristics of the atom in the specimen are emitted. The

analysis provides elemental maps of some important elements (calcium, phosphorus, fluorine and strontium) within the specimen (62). SEM is suitable for bone surface morphology, reflecting the local metabolic activity of bone cells (63). Age-related changes in cortical bone microarchitecture, such as osteon development, lamellar apposition rate and Haversian canal morphological changes, can easily be studied using SEM. (64). Overall, SEM provides important quantitative information of the bone that can be related to the lamellar topography, canalicular network, osteocyte density, resorption spaces and microcracks. However, SEM techniques have some methodological difficulties, such as the need for dehydration, acid etching, and conductive coating of the specimen before analysis.