New bone formation starts as osteoblasts begin the deposition of an organic matrix, 90% of which is constituted of type I collagen [16]. The collagen backbone consists of alpha chains of repetitive amino acid triplets that raise absorption subbands beneath amide I, II, III, A and B regions of the FTIR spectrum [9]. As expected, these different absorption bands are also observed in the absorption spectra of tissues in the entire osteoid and cortex (Fig. 2). In addition, the proline amino acid in collagen raises an absorption band in the FTIR spectrum at 1338 cm−1 which is specific to collagen type I [38]. The linear augmentation of the collagen-absorption band at 1338 cm−1 in the spectra of tissues inside the osteoid demonstrates a gradual accumulation of collagen. This deposition is mainly completed by the end of the growth zone. Next, the single alpha-chains fold into triple helices that are stabilized by hydrogen bonds provoking absorption in the infrared spectrum at the wavenumbers at around 3070 cm−1 , in the amide-B-spectral region [10]. In this study, this absorption band at 3070 cm−1, appears in the spectrum of tissues at the border with osteoblast layer and continues to increase for the tissues situated inside the osteoid with the tissue distance from the osteoblast border (Fig. 2), which indicates that the triple helix is consolidated immediately when the collagen is secreted.

The triple-helical collagen fibrils are then assembled into collagen fibers that are stabilized by the formation of covalent intermolecular cross-links. This phenomenon is called “collagen maturation”. First, three different immature-divalent cross-links are created: dehydro-hydro-lysinonor-leucine (deH-HLNL), dehydro-dihydro-lysinonor-leucine (deH-DHLNL), dehydro-lysinonor-leucine (deH-LNL) depending on the molecules involved in the linking [30]. In the mineralizing front and in the near cortex, because collagen accumulation is accomplished, the deH-DHLNL-subband-absorption diminution is due to the deH-DHLNL cross-link diminution only, which confirms the immature deH-DHLNL cross-links predominance in young bone [27].

Second, the immature-divalent cross-links are further assembled into mature trivalent cross-links: Hydroxylysyl-pyridinoline (Pyr), pyrroline (Prl), lysyl-pyridinoline (DPD) and deoxy-pyrrololine (d-Prl) [27, 31]. By consequence, immature deH-DHLNL cross-links should diminish for the profit of mature Pyr cross-link, which happens in the bone cortex of this study. Because the collagen accumulation is accomplished at the end of the osteoid, the absorption variation of Pyr-, DPD- and deH-DHLNL-subbands are proportional to the amount of created Pyr, DPD and reduced deH-DHLNL cross-links in cortex. In addition, the linear augmentation of XLR-index magnitude proves that deH-DHLNL cross-link is reduced in Pyr cross-link in the early cortex. Besides, the Pyr-subband-absorption variation observed in this study was also observed by Imbert et al. for cancellous vertebrae of sheep imaged with AFM-IR technique [20]. However, in the growth zone, Pyr-; DPD- and deH-DHLNL-subband intensity, and XLR-index magnitude variations are influenced by the collagen accumulation which is not completed in this region making it impossible to determine the absolute Pyr-quantity variation in this zone. Contrary to the study by Imbert et al. [20], who found that XLR-index magnitude is constant with the distance from the trabecular border, the XLR-index magnitude in this study increases in the cortex after the osteoid and then is constant in deep cortex. Our finding is in accordance with the study of human’s iliac crest bone by Faibish et al. [11]. This demonstrates a rapid collagen maturation in the cortex adjacent to the osteoid whereas no collagen maturation was observed in the deep cortex. The Pyr/DPD-index magnitude increases constantly in the early cortex until the deep cortex which indicates that Pyr-cross-link formation is higher than DPD-cross-link formation in bone as Viguet-Carrin et al. propose [42].

During mineralization, the osteoblasts produce matrix vesicles transporting a precipitated constituent of calcium phosphate into the collagen scaffold [18, 25]. After its secretion, the calcium phosphates are transformed into amorphous calcium phosphates (ACP) which transform into octacalcium phosphate (OCP) and gradually into hydroxylapatite (HA) [41]. The phosphate in presence of crystalline fields provokes an absorption band in the infrared spectrum between 950 and 960 cm−1, i.e. in the \(\nu_1\) \(}_4^\) spectral region. In this study, the mineral accumulation, represented by integrated intensity of the area under the \(\nu_1\) \(}_4^\) region, presents a sigmoid variation with a strong slope in the mineralizing front and adjacent cortex to osteoid which indicates that the collagen mineralization appears only in mineralizing front and finishes further away in the cortex. The mineralizing front feature seems to be analogous to the mineralizing front observed in electron microscopy [25]. The linear augmentation of integrated value of the area under the \(\nu _1\) \(}_4^\) region, demonstrates a progressive collagen mineralization in the mineralizing front of the osteoid and in the cortex adjacent to osteoid. In addition, at the end of the osteoid, the mineral content reaches 50% of its total amount indicating that the mineralizing front corresponds to the primary mineralization whereas the adjacent cortex corresponds to the secondary mineralization [2].

The transformation of ACP into HA provokes a progressive shift of the absorption band at 950 cm−1 to the right until it reaches 961 cm−1 [17, 36]. In this study, this absorption-band shift is observed for tissues inside the osteoid and continue to grow in the cortex adjacent to osteoid, which signifies a progressive formation of the HA, beginning slightly before the mineralizing front, and continuing to form in the mineralizing front and adjacent cortex. In addition, in osteoid, the evolution of the shape of the spectrum in the \(\nu_3\) \(}_4^\) spectral region corresponds to the ones of autocatalytic conversion of amorphous calcium phosphate to poorly crystalline HA [32], which confirms the transformation into HA begining in osteoid. The formation of HA, in mineralizing front, is also observed by studying the absorption band due to \(O-H\) at 3570 cm−1 because O–H-stretching vibration at this wavenumber is unique to crystalline hydroxylapatite [40]. In this study, the shoulder at around 3570 cm−1 is observed in mineralizing fronts which demonstrates that the formation of crystalline HA occurs in the mineralizing front of osteoid. This coincides with the beginning of the absorption band peak shifting observed in \(\nu_1\) \(}_4^\) region

After hydroxyl-carbonate-apatite formation, mineral-maturity index (CM) starts to increase [44] i.e. the hydrated-surface layer covering the apatitic crystal, called non-apatitic environment, is progressively transformed into hydroxyl-carbonate apatite reducing the amount of non-apatitic \(}_4^\) [43]. In this study, the variation of CM-index magnitude increases in the early cortex, and it reaches a plateau in the deep cortex indicating that bone-mineral maturation starts just after the osteoid when the HA is formed and continues to mature in the near cortex until the deep cortex. This variation is in accordance with studies about iliac crest bone in previous literature [12]. However, in the mineralizing fronts, CM-index magnitude is constant indicating that the mineral maturation may not begin yet. However, inside the osteoid and cortex adjacent to the osteoid, the mineral is still accumulating which creates non-apatitic \(}_4^\) ions leading to an augmentation of the subband intensity at 1110 cm−1 and by consequence to a diminution of CM value preventing the use of CM index for any mineral-maturity characterization in osteoid.

Because the non-apatitic domains are transformed progressively in well-crystallized apatite, during the mineral maturation, the crystallinity should increase during this transformation. The crystallinity of bone mineral was proved to correlate with the variation of XST index, i.e. of the proportion of the subband intensity at 1030 cm−1 (subband found in well-ordered-crystallized material) relative to the one at 1020 cm−1 (subband found in poorly crystalline material) [33]. XST is a different characteristic than CM [12]. In this study, in the middle and deep cortex area (after 58  μm), XST index magnitudes increase first and then reache a plateau, indicating that crystallinity first increases and then remains constant in the deep cortex. This XST index variations correlate with the variations in cortex found for human iliac crest bone in the literature [7]. However the XST index describes the crystallinity of HA only if other components have not affected this index, which is the case in the middle and deep cortex where the transformation into HA and the phosphate accumulation is finished. This is not the case in osteoid and in its adjacent cortex. In these regions, the phosphate is still accumulating and undergoes several transformations leading to the creation of additional octacalcium and tetracalcium phosphate which will be transformed later into HA. These created calcium phosphates increase the absorption band at 1020 cm−1 of the XST index which could lead to the diminishion of XST observed in the cortex adjacent to osteoid whereas the crystallinity may not diminish [6].

During the maturation of the HA crystal several ion substitutions occur: substitution by a carbonate or by an acid phosphate [2]. In bone, predominant B-type-carbonate substitutions disturb the crystal shape and biomechanical properties [45] and increase the crystal solubility due to a weaker \(}}_3\) bond than \(}}_4\) bond [37]. Also, in this study, the amounts of B-type-carbonate substitute were superior to the A-type-carbonate substitute in the deep cortex. In the osteoid, the amounts of B- and A-type-carbonate substitutions increase slightly in the mineralizing fronts, demonstrating that carbonate substitution could already begin in osteoid. However, stronger substitutions are observed in the near cortex right after osteoid, indicating that the B- and A-type-carbonate substitutions occur mostly in the new cortex. In the deep cortex, both B- and A-type-subband intensities are almost constant. These results are in accordance with previous reports found in the literature [19, 28].

Acid phosphates (\(}_4^\)) are found in bone apatite substituting a phosphate ion \(}_4^\) in HA [43]. In this study, APS-index magnitudes decrease strongly in the mineralizing fronts and in the early cortex indicating a stronger substitution by acid phosphate in these areas than in the deep cortex which confirms that the new-formed bones, presents a higher APS-index value than older bones [39].

In summary, it was demonstrated in this study that collagen aggregates in osteoid early before the mineralization initiation. The collagen aggregation is finished in the middle of osteoid, slightly after the mineralizing front. This temporal difference has also been referred to as mineralization lag time [22]. During collagen accumulation, collagen also starts to mature. However, the mineralization begins before than the collagen reaches its full maturity. The phosphates reach half of its final content by the end of the osteoid coinciding with the end of collagen accumulation and continue to accumulate in the cortex adjacent to osteoid until further away in the cortex. However, the mineral maturation appends right after the osteoid when the phosphate reaches its half content and ends further away in the deep cortex. When the phosphate begins its accumulation, it immediately undergoes carbonate and acid phosphate substitutions. The acid phosphate substitution diminishes as the crystal develops. In contrast, the amount of carbonate substitute increases with the accumulation of phosphate and ends in the deep cortex. These different variations and their relations presented in this paper confirm that mineralization and collagen deposition occur at different time points and that the mineralization begins when the collagen is not completely mature. This new information provides a better understanding of bone remodelling in human-mandibular bone which provides a base for studying bone pathologies caused by flaws in bone remodelling. In the future, differences in osteoid-mineralization patterns for non-healthy bones should be investigated to provide an explanation of the differences between bone types and pathologies.