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-/ν4PO43- or the ratio of
ν1CO32-/ν1PO43- 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.