Biomineralization and Biointerfaces

Osteoblast-to-Osteocyte Transformation
Osteoblast-to-osteocyte transformation is investigated using sodium hypochlorite deproteinisation, enabling SEM imaging of isolated bone mineral. This reveals the evolution of the extracellular matrix (ECM), where partially embedded osteoblastic-osteocytes indicate tissue age and maturation. In regions of new bone formation near osteoclastic resorption sites, the bone surface and osteocyte lacuna floor exhibit repeating motifs (~1.5–2 µm long, ~1 µm wide), composed of plate-like bone apatite crystals (~20–25 nm thick) aligned with type-I collagen fibrils (Figure 2).

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Osteocytes and Implants
Although optical microscopy (e.g., histology) reveals direct bone-implant contact, osteocyte interactions with implants remain difficult to observe. Resin cast etching exposes the osteocyte lacuno-canalicular network and reveals osteocyte attachment to implant surfaces via canaliculi (Figure 3). High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) provides detailed imaging of canaliculi near bone-anchored metal implants.

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Resin cast etching also enables visualisation of bacterial invasion in osteonecrosis, demonstrating bone microstructural degradation due to infection (Figure 4).

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Backscattered electron scanning electron microscopy (BSE-SEM) reveals bone microstructure, making it a valuable tool for assessing bone ingrowth into 3D printed metal implants (Figure 5).

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Elemental analysis (point measurements and mapping) using EDX can be performed in both SEM or TEM (operated in the scanning transmission electron microscopy mode (STEM), which enables high resolution (nanoscale) analysis.

Example 1: SEM-EDX shows compositional differences between the two types of mineral nodules observed in micropetrosis; rhomboidal nodules of magnesium whitlockite and spherical nodules of carbonated apatite (Figure 6).

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Example 2: STEM-EDX of mineral nodules within osteocyte lacunae and the surrounding bone (Figure 7).

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Example 3: SEM-EDX shows that in rodents (e.g., rat) pigmented enamel (PIE) of incisor teeth is iron-rich while the unpigmented enamel (UME) of molar teeth is magnesium-rich. Scanning electron microscopy reveals the microstructural differences between pigmented and unpigmented enamel (Figure 8).

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Example 4: STEM-EDX of the bone-implant interface reveals a chemically graded interface with gradual intermixing of titanium and calcium signals, where titanium–oxygen overlap confirms titanium oxide and calcium–phosphorus overlap confirms the presence of bone (Figure 9).

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Spectroscopic tomography, a 4D technique combining chemical and spatial data, enables differentiation between intrafibrillar and extrafibrillar mineral, with extrafibrillar mineral displaying higher calcium and phosphorus content (Figure 10).

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Micro-CT can be used to analyse growth and development. Conditions such as congenic leptin receptor deficiency are associated with discrepancies in cranial suture (Figure 11) and femur (Figure 12) development.

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Combining micro-CT, histology, polarised light microscopy (PLM), BSE-SEM, and HAADF-STEM allows investigation of bone ingrowth and remodelling within calcium phosphate biomaterials (Figure 13).

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Bone micro-porosity can be characterised using BSE-SEM to distinguish vascular spaces from osteocyte lacunae (Figure 14).

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Particulate materials, such as deproteinised bovine bone mineral, exhibit higher crystallinity than native bone. Analysis using backscattered electron scanning electron microscopy, energy dispersive X-ray spectroscopy, Raman spectroscopy, and X-ray diffraction confirms loss of carbonate ions due to the manufacturing process (Figure 15).

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Raman spectroscopy differentiates bone mineral from synthetic hydroxy(l)apatite. Compared to synthetic HAp, bone generates markedly higher background fluorescence due to the organic matrix (Figure 16).

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In rodents, pigmented enamel has higher crystallinity and hydroxyl ion content than unpigmented enamel, as shown by Raman spectroscopy (FIgure 17).

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Raman spectroscopy of mineralised bacterial biofilms on dental implants reveals carbonated apatite in bacterial regions, while octacalcium phosphate and whitlockite are found where bacteria are absent, indicating microenvironmental variations (Figure 18).

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