*3.2. Scanning Electron Microscopy*

SEM examination of human spongiosa that underwent all stages of the Lyoplast® bioimplant production process, including ultrasound treatment of the biomaterial, demineralization, lyophilization, and γ-sterilization, showed complete preservation of the original trabecular bone architectonics. According to the results of the SEM, the completed biopolymer product is a porous three-dimensional (3D) matrix with a hierarchical structure of pores of various calibers (300–800 μm), free of cellular and bone marrow components. Trabeculae of lamellar bone are visualized on the image; their contours are precise and interconnected to form fine pores. Similarly, these fine pores do not contain cellular and bone marrow components. The 3D photos demonstrate a self-similar hierarchical architecture of the human spongiosa organization (Figure 2).

**Figure 2.** Architectonics of demineralized lyophilized human spongiosa (SEM). Trabecular architecture: (**A**) cross section of trabeculae (marked by an arrow), ×100; internal structure of the trabeculae (**B**) ×500.

#### *3.3. Micro-Computed Tomography*

Non-destructive microstructural analysis of the Lyoplast® human spongiosa bioimplant using computed microtomography confirmed the preservation of spongy bone's trabecular architectonics and porous microstructure with a pore caliber of 300 to 800 μm. Three-dimensional reconstruction of bone tissue samples using a resolution of 8.6 μm/pixel and a voltage of 80 kV made it possible to visualize the surface of the pores, free from cellular debris and fatty components of the bone marrow (Figure 3).

**Figure 3.** Architectonics of demineralized lyophilized human spongiosa (Micro-CT). Trabecular architecture: (80 kV, 8.6 μm/pixel; 1081 projections; 0.5 s exposition time).

Using a resolution of 1.1 μm/pixel and a voltage of 80 kV confirmed the hierarchical bone tissue architectonics and allowed us to visualize osteocyte lacunae in the intrinsic structure of bone trabeculae. The average caliber of osteocyte lacunae was 10–30 μm. The investigation also confirmed the absence of a cellular component in the lacunae. In addition, three-dimensional image inversion allowed a detailed analysis of the number and condition of osteocyte lacunae. (Figure 4). An essential advantage of micro-computed tomography compared to scanning electron microscopy (SEM) is the combination of high-throughput fashion and ultra-high resolution, making it possible to simultaneously visualize a significant number of osteocyte lacunae while maintaining high image quality.

**Figure 4.** Architectonics of demineralized lyophilized human spongiosa (Micro-CT). Trabecular architecture: (80 kV, 8.6 μm/pixel; 1081 projections; 0.5 s exposition time) (**a**)—visualization of the osteocyte lacunae; (**b**,**c**)—image inversion and osteocyte lacunae detection).
