Figure 3. a) A macroscopic-to-microscopic view of cancellous and cortical bone. B) Schematic diagram of the triple helix structure of self-assembled collagen fibers in bone.[19]Copyright 2018, Elsevier.
Biomineralization Mechanism
Collagen
Collagen is present in most biomineralized tissues in vertebrates, and collagen provides nucleation and crystallization sites for minerals (Figure 3a ). Collagen fibers are divided into three types, such types Ⅰ, Ⅱ, and Ⅲ, which consist of glycine-X-Y repetitive sequences in collagen fibers (Figure 3b ) where X is proline or hydroxyproline and Y is lysine or hydroxylysine. Both of which can be detected in Raman spectra. Amide I (1675 cm-1) and Amide III (1272 cm-1) bands did not change with increasing glycosylation, indicating the conserved triple helix structure of collagen fibers.[146] However, significant changes in the proline may affect bone toughness. In addition, the tissues created by longitudinal fiber stacking have more tensile properties than those created by transverse fiber stacking.[147] The involvement of longer collagen fibers also enhances the toughness of the bone.[148] This means that when the ratio of ν1PO43-/Amide III (960/1272 cm-1) increases, the bones become more fragile (Table 2 ).
Amide I is a characteristic peak of the collagen secondary structure, not the collagen crosslink content.[148] The secondary structure formed by less collagen cross-linking can directly show the Raman characteristic peak. It is often used as an internal standard for the relative quantification of other substances.[149] Due to the different content of collagen in different biomineralized tissues, Raman mapping of Amide I and Amide III was also used to distinguish between the two, such as cartilage and cortical bone, and so on.[150–152]
Hydroxyapatite
Hydroxyapatite is an important component of bones and teeth in the human body. The c-axis is the direction of hydroxyapatite crystal growth with an aspect ratio of up to 1000 (Figure 4a ). The main mineral component in teeth and bones is hydroxyapatite, which exhibits ν1 PO43-, ν2 PO43-, ν3 PO43-, and ν4 PO43- fundamental frequency modes in the Raman spectrum (Figure 4b ). The orientation of this hydroxyapatite affects the characteristic peaks of ν1 PO43- in the Raman spectra of the enamel.[122,122,149] Hydroxyapatite is not directly produced during the maturation of enamel or bone.[153–155] Rather, nucleation and crystallization of amorphous calcium phosphate (ACP) on collagen fibers. Over time, the crystalline phase transforms into calcium phosphate (OCP), eventually forming mature hydroxyapatite crystals.[32,155–158] During this process, ν1 PO43- begins to red-shift from 950 to 960 cm-1 (Table 2 ).
During the maturation of the enamel, ions (Mg2+, Cl-, F-, OH-, CO32-) are constantly embedded in the lattice of hydroxyapatite crystals.[159–162]Ionic doping is generally achieved by replacing hydroxyl groups with hydroxyapatite. The intensity or frequency of phosphate and carbonate vibrational peaks can be used to quantify the type and extent of halogen ion substitution.[163,164] Then, ν4 PO43- in hydroxyapatite crystals, unlike ν1PO43-, is not affected by crystal orientation, and its half-peak width is often used to analyze crystal crystallinity or is used as an internal reference.[165–167] The half-peak width of ν1 PO43-, the ratio of ν1CO32-4PO43- or the ratio of ν1CO32-1PO43- have also been studied as indicators of the degree of hydroxyapatite crystallization.[150,165–167] Interestingly, the higher crystallinity of hydroxyapatite crystals leads to an increase in hardness and a decrease in toughness.