*3.4. Raman Spectroscopy*

Raman spectroscopy allowed us to obtain detailed information about spectral contour decomposition of demineralized spongiosa samples using a Gaussian function as a trial (Figure 5). The mean value of the coefficient of correlation between the recovered and input spectrum (R2) in the region of 750–2050 cm−<sup>1</sup> was 0.99, indicating near-perfect agreement.

**Figure 5.** Spectral contour decomposition of demineralized spongiosa samples. The solid line-original Raman spectrum; the dashed lines-the Raman lines after separation.

We have established no mineral components in the demineralized spongiosa, as indicated by the disappearance of the Raman line at 956 cm−1, corresponding to *PO*43−(ν1). As can be seen in Figure 3, the demineralized spongiosa lacks fat, as indicated by the absence of an intense Raman line at 1307 cm−<sup>1</sup> (lipids). At the same time, the preservation of the organic matrix is observed, as indicated by the presence of Raman lines at 850 cm−<sup>1</sup> (proline), 1238 cm<sup>−</sup>1–1272 cm−<sup>1</sup> (Amide III), 1450 cm−<sup>1</sup> (proline), 1167 cm−<sup>1</sup> (glycosaminoglycans), and 1660 11 cm−<sup>1</sup> (Amide I). Collagen is the main protein component of bone tissue, and it forms the fibrillar framework of the bone matrix. The amino acid sequence

of collagen is notably rich in proline, about half of which is hydroxylated during collagen breakdown to form hydroxyproline.

#### *3.5. Mass Spectroscopy*

Proteomic analysis of the demineralized human spongiosa demonstrated the presence of collagenous and extracellular bone matrix proteins. The ability of these proteins to stimulate and inhibit cell adhesion, proliferation, and differentiation is noteworthy. We identified five main types of collagen (I, IV, VI, XII, XIV), fibronectin, vitronectin, osteopontin, matrix Gla-protein, along with TGF-β mimecan, decorin, and other proteins in the human spongiosa organic matrix. A list of these proteins, their localization, and their mass are presented in Table 1.

**Table 1.** List of organic components identified in the analysis of demineralized human spongiosa Lyoplast®.


We assign properties and functions of the identified proteins according to Baghy et al. [25–28]. In summary, collagen acts as a cell-binding protein that performs an adhesive function by integrating collagen bundles, a major component of the extracellular matrix. Bone, basal membrane, and soft tissue collagens are found in human spongiosa. Fibronectins bind cell surfaces and compounds, including collagen, fibrin, heparin, DNA, and actin. Fibronectins are involved in cell adhesion, cell motility, opsonization, wound healing, and maintenance of cell shape. They also participate in the regulation of type I collagen deposition by osteoblasts. Vitronectin is a glycoprotein of the hemopexin family, which is abundant in serum and the extracellular matrix of bone tissue. It is involved in fibrinolysis, mediates cell adhesion and migration, and binds glycosaminoglycans, collagen, and plasminogen. These three organic substances (collagen, fibronectin, vitronectin) are widely used in biotechnology to create the cytoadhesive surface of the culture plate.

Mimecan, or osteoglycin, is a small proteoglycan rich in leucine, important for collagen fibrillogenesis. Decorin, lumican, and biglican are small proteoglycans of the extracellular matrix that bind to fibronectin, inhibit cell adhesion, attach to tumor growth factor, and reduce tumor cells' mitogenic activity. They play a regulatory role in connective tissue development and repair processes. TGF-β1 is a secreted protein that performs many cellular functions, including control of cell growth, cell proliferation, cell differentiation,

and apoptosis. Osteopontin is involved in cell proliferation, migration, and adhesion, including bone marrow mesenchymal stem cells, hematopoietic stem cells, osteoclasts, and osteoblasts. Osteonectin is a bone tissue glycoprotein that binds calcium. It is released by osteoblasts during bone formation, initiating mineralization and promoting the formation of mineral crystals. Osteonectin also has an affinity for collagen. Finally, bone sialoprotein is a critical component with a high sialic acid content of the extracellular bone matrix, constituting approximately 8% of all non-collagenous proteins. It has the function of forming the hydroxyapatite core during bone mineralization. Matrix Gla-protein associates with the organic matrix of bone and cartilage and acts as an inhibitor of bone formation, thereby playing an essential role in bone mineralization but acting as a mineralization inhibitor in cartilage and vessels. Finally, the extracellular matrix protein tenascin is involved in the control of migrating neurons and axons during neuronal development, synaptic plasticity, and regeneration. Its role in osteoblasts differentiation is unknown. This comprehensive analysis of protein constituents complements the results of Raman spectroscopy and provides a broader understanding of the biochemical composition of the organic matter of human spongiosa Lyoplast®.

#### *3.6. Obtaining a Line of Human Juvenile Chondroblasts and Creating a Cell-Tissue Graft*

Observations of the native culture showed that cells had excellent adhesive properties. On the first day in culture, most chondroblasts from the suspension were deposited from the medium to the bottom of the plate, where they attached and spread out. They took on an elongated shape with a well-defined boundary, connecting through 3–5 processes. The cytoplasm contained many vacuoles in the peripheral zone. The nucleus was oval shaped, usually located in the center of the cell, and contained 1–3 nuclei. The number of cells gradually increased during cultivation, forming a uniform monolayer. When the culture reached 80% confluence, the chondroblasts adhered tightly to each other, and there were practically no areas free of cells. At this stage, cell transplantation was performed using the standard method. A qualitative culture assessment was carried out using cultural and morphological methods in the fourth passage. Our earlier preclinical animal studies, using combined cell-tissue grafts based on rabbit allogenic demineralized spongiosa and rabbit rib cartilage cell culture, showed the healing of the animal joint's simulated bone and cartilage defects. Finally, organotypic hyaline cartilage tissue was revealed [19], and it is promising for translational use of Lyoplast® human spongiosa. The unique structure and composition of the demineralized human spongiosa allowed us to use it as an effective bioscaffold for creating the cell-tissue graft.

The findings reported by Doran and his study group members in 2021 [29] also demonstrated the efficiency of using cartilaginous differon cells for articular cartilage tissue restoration. Obtained results demonstrate the prospect for further use of demineralized human spongiosa for articular hyaline cartilage defects repair. Obtaining such cell-tissue grafts is relatively simple and does not require complex bioreactor systems.

Cell cultures stained with hematoxylin and eosin showed the geometric pattern typical of cartilage differon cells. The cells were aligned, forming concentric and ellipsoidal figures resembling osteons and insertion plates of compact bone tissue. The presence of polygonal cells was noted. The cytolemma of chondroblasts in culture was smooth, and the weakly oxyphilic cytoplasm contained vacuoles. Nuclei of regular round shape with a smooth envelope were located mainly in the center. Chromatin in the form of fine granularity was located diffusely in the nuclei. The large processes were shortened, and the intercellular substance was visualized as translucent layers (Figure 6a). The fluorescence microscopy with a Live/Dead® fluorophore kit revealed 93% viable cells (Figure 6b).

(**a**) (**b**)

**Figure 6.** Human juvenile chondroblast culture: (**a**) hematoxylin and eosin staining. Light microscopy, ×100; (**b**) live chondroblasts exhibiting bright green glow. Fluorescence microscopy. Staining with Live/Dead® fluorophores, <sup>×</sup>100.

Upon examining living and damaged cells populated on a 3D carrier made of Lyoplast® human spongiosa using the Live/Dead® kit, we detected viable cells with bright green coloration and damaged or dead cells with a bright red nucleus (Figure 7a). On closer examination, the cells on the surface of the trabeculae were evenly distributed throughout the scaffold depth, and the shape was polygonal (elongated, triangular, rounded). We visualized the outgrowths with which the chondroblasts were connected, creating a uniform monolayer, which was indicative of good cell adhesion to this material. When the cell-tissue graft was examined using SEM, we saw adsorption of proteins on the trabeculae surface, where cells of elongated shape were connected by outgrowths (Figure 7b).

**Figure 7.** Demineralized spongiosa using the Lyoplast® technology with populated chondroblasts: (**a**) populated viable chondroblasts on the surface of the biocarrier (arrows indicate attached cells). Live/Dead® fluorophore staining. Fluorescence microscopy, <sup>×</sup>100; (**b**) cells inhabiting the surface of trabeculae are marked by arrows. SEM, ×100.

#### *3.7. MTT Assay of the Demineralized Human Spongiosa MTT Test Results*

MTT Assay of the Demineralized Human Spongiosa MTT Test Results Are Presented in Table 2 and Figure 8.


**Table 2.** MTT test. Indicators of optical density in experimental and control wells.

Using MTT test (48 h treatment), it was found that for the experimental group (Cells + Spong and controls (Cells only)), the optical density was 0.491 ± 0.023 and 0.512 ± 0.003, correspondingly. Statistical analyses revealed that no significant statistical differences in cell viability (*p* = 0.513) were observed between control cells (Cells only) and cells grown in the presence of the investigated biomaterial (Cells + Spong).

Thus, the investigated biomaterial—demineralized human spongiosa Lyoplast®—is not cytotoxic.

**Figure 8.** MTT test. No difference in cell viability between control (Cells only) and probe (Cells + Spong) was detected by MTT test.

#### **4. Conclusions**

Microstructural analysis of the demineralized human spongiosa convincingly demonstrates its preserved hierarchical porous structure. Pores of various configurations and calibers are entirely free of cellular debris and bone marrow components. Furthermore, the proteomic analysis confirmed the complete preservation of collagen and other extracellular matrix proteins. These proteins play a crucial role in inhibiting and stimulating cell adhesion, proliferation, and differentiation. We revealed the adhesion of chondroblast cell cultures in vitro without any evidence of cytotoxicity. Revealed features of the demineralized human spongiosa create optimal conditions for hyaline articular cartilage and subchondral bone regeneration. Thus, according to the study results, demineralized human spongiosa Lyoplast® can be effectively used as the bioactive scaffold for articular hyaline cartilage tissue engineering. For the future perspective, the identified microstructural and biochemical features of the demineralized human spongiosa can be considered in 3D bioprinting and creating tissue-engineering constructs of the cartilage tissue.

#### **5. Patents**

Patent RF № 2170016 from 17.02.1999 "Method of saturation of bone spongy tissue grafts with medication" Volova L.T., Kirilenko A.G., Uvarovsky B.B.

Patent RF № 2156139 from 15.03.1999 "Method of sterilization of lyophilized bone transplants" Volova L.T., Kirilenko A.G., Uvarovsky B.B.

Patent RF № 99108699 from 21.04.1999 "Method of bone marrow removal from cancellous bone grafts" Volova L.T., Kirilenko A.G.

Patent RF № 2366173 of 15.05.2008. "Method of manufacturing large-block lyophilized bone implants" Volova L.T.

**Author Contributions:** Production of demineralized lyophilized spongiosis Lyoplast®, interpreting the received experimental results, I.L.T.; Conception, methodology, research design, analysis of results, conclusions, original writing, L.T.V.; project management, review preparation, editing, revision, A.V.K.; staging and conducting in vitro studies, drafting, visualization, E.I.P.; construction of experiments methodology using Raman spectroscopy and processing of the obtained experimental data, E.V.T.; conducting research using fluorescence microscopy, interpretation of the results obtained, visualization, V.V.B.; carrying out the experimental study using the Raman spectroscopy method, processing and interpreting the received results of the study, finalizing the article, P.E.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki. The protocol was approved by the Ethics Committee (extract 20.01.2021no.215 of minutes of the meeting of the Committee on Bioethics of Samara State Medical University).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available on request due to restrictions eg privacy and ethical.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

