**1. Introduction**

Current treatment options for degenerative bone and cartilage tissue pathology aim to enhance post-traumatic and post-operative defects regeneration using various biological or synthetic products.

Biodegradable scaffolds, including calcium phosphate, aerogels [1–6], autologous [7–9], allogeneic [10–12], or xenogeneic grafts [13–17] demonstrate significant efficiency as bone substitute materials. Ideally, biocompatible materials' resorption rate coincides with the formation rate of the new organotypic tissue. Allogeneic products incorporate identical structural and biological components and provide optimal conditions for genetically programed physiological regeneration in the human body [10,18]. Original technology of the human bone tissue products manufacturing developed at Samara Tissue Bank at Samara State Medical University has been successfully applied in bone tissue repair for more than twenty years. This technology provides thorough mechanical cleaning and complete removal of the antigenic components from human spongiosa while preserving its biological activity [18,19]. Microstructural and biochemical properties of the natural biopolymers play a crucial role in the regeneration process and directly depend on the manufacturing technology.

This study aims to investigate the microstructure and biocompatibility of the novel biopolymer material from demineralized human spongiosa.

**Citation:** Tsiklin, I.L.; Pugachev, E.I.; Kolsanov, A.V.; Timchenko, E.V.; Boltovskaya, V.V.; Timchenko, P.E.; Volova, L.T. Biopolymer Material from Human Spongiosa for Regenerative Medicine Application. *Polymers* **2022**, *14*, 941. https:// doi.org/10.3390/polym14050941

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 20 January 2022 Accepted: 22 February 2022 Published: 26 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### **2. Materials and Methods**

#### *2.1. Manufacturing and Characterizing Materials*

The biopolymer Lyoplast® analyzed in this study is lyophilized demineralized human spongiosa manufactured at the Samara tissue bank at the "BioTech" Biotechnology Center, Samara State Medical University (RF patent No. 2366173 of 15.05.2008; certificate of conformity ISO 13485:2016, reg. No. RU CMS-RU.PT02.00115; certificate ISO 9001:2015, reg. No. TIC 15 100 159171) (Figure 1).

**Figure 1.** Samples of demineralized lyophilized human spongiosa Lyoplast®.

Experimental samples of Lyoplast® material underwent compulsory low-frequency ultrasonic treatment using ultrasonic bath "Sapphire" TTC (RMD), (Sapphire LTD, Moscow, Russia with a frequency of 24–40 kHz.

Lyophilization of the material (vacuum drying by sublimation) was performed using a sublimation unit ALPHA2-4LSC (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).

Demineralization of human spongiosa was carried out in a weak HCl solution. Hermetically packaged lyophilized product was then sterilized with gamma rays using a certified GU-200 M (NIIP Joint-Stock Company, Moscow, Russia).

The residual content of lipids in the biomaterial was estimated using a spectrophotometer (SF-56 "Lomo-Spektr", St. Petersburg, Russia). Finally, the humidity of the product was determined using a thermogravimetric infrared moisture meter (Sartorius-MA-150, Malente, Germany).

The study was carried out using physical, chemical, biological, and cultural methods.

#### *2.2. Scanning Electron Microscopy (SEM)*

The samples were examined using a JEOLJSM-6390 A Analysis Station SEM (Tokyo, Japan). Bioimplant samples were washed and fixed with a 2.5% aqueous solution of glutaric aldehyde. After that, they were spiked with ethanol of increasing concentration, followed by drying at room temperature for 24 h. Immediately before the study, the biomaterial was sprayed with gold or carbon to improve the surface electrical conductivity required for SEM.

#### *2.3. Micro-Computed Tomography (Micro-CT)*

Micro-CT scanning of the Lyoplast® lyophilized allogeneic spongiosa samples was performed in Laboratory of Microanalysis in Skolkovo Technopark (Moscow, Russia) using high-resolution 3D X-ray microscope VersaXRM-500 (Xradia, Inc. Pleasanton, CA, USA) with voltage range 30–160 kV, maximum power 10 W, 360◦ rotation, and maximum spatial resolution < 0.7–1 μm (True Spatial Resolution™). At the first stage, the scanning of the sample was performed using a resolution of 8.6 μm/pixel at a voltage of 80 kV with a set of

1081 projections and 0.5 s exposition ti0me. Next, a region of interest (ROI), including bone trabeculae [20] was selected and scanned with a resolution of 1.1 μm/pixel at a voltage of 80 kV with 1441 projections and 0.5 s exposition time. The obtained data were reconstructed with the Filtered Back Projection method using the XRM Reconstructor software. Computed microtomography data were saved in TXRM and DICOM formats, and 3D models of the sample structure were saved in TXM and TIFF formats.

#### *2.4. Raman Spectroscopy (Raman Spectroscopy)*

This research was performed at the Department of Laser and Biotechnical Systems of Samara National Research University. Spectral characteristics of lyophilized, demineralized human spongiosa Lyoplast® were studied using an experimental setup that included a highresolution digital spectrometer Shamrock SR-303I (Oxford Instruments PLC, Abingdon, UK) with a built-in cooling chamber AndorDV420A-OE (Oxford Instruments PLC, Abingdon, UK) and an RPB785 fiber-optic probe combined with a laser module LuxxMasterLML-785.0RB-04(Laser Components Germany GmbH, Olching, Germany), all under the control of a PC workstation. This spectrograph provided 0.15 nm wavelength image resolution with low intrinsic noise. To exclude the autofluorescence contribution in the Raman spectra, we used a method for subtracting the fluorescence component of the polynomial approximation with additional filtration of random noise effects. In this work, the Raman spectra were analyzed in 350–2200 cm<sup>−</sup>1. The laser power of 400 mW was applied for 30 s exposure time, without evident degradation of the samples. Raman spectra were registered using an optical probe, which was placed above the object at a distance of 7 mm. We used the method of spectral contour fitting and deconvolution of the Gaussian function in the software environment MagicPlotPro 2.7.2. Thus, we conducted a non-linear regression analysis of Raman spectra to decompose the signal into spectral lines [21–24].
