*2.5. Mass Spectroscopy (Proteomic Analysis)*

Samples of lyophilized, demineralized spongiosa were subjected to heat treatment (100 ◦C, 5 min) in a medium containing 2% SDS (sodium dodecyl sulfate) and 5% mercaptoethanol, centrifuged, and separated in a 10% polyacrylamide gel in the presence of SDS. After electrophoresis, the gel was stained with Coomassie G250 and cut into fragments according to the visually detectable fractions. For qualitative identification of tightly bound proteins, gel fragment samples were washed with ammonium hydrogen carbonate and acetonitrile solution (1:1) at 50 ◦C, dehydrated in 100% acetonitrile, and then treated with trypsin solution in 50 mM ammonium hydrogen carbonate. The reaction was stopped with the addition of 0.1% trifluoroacetic acid, and the released peptides were extracted from the gel by placement in an ultrasonic bath and separated by high-performance liquid chromatography (Dionex Ultimate 3000, Country) using an AcclaimPepMap C18 analytical column (2 μm, 100 Å, 75 μm × 15 cm) (Thermo Scientific). Mass spectra of the eluant were obtained on a maXis Impact mass spectrometer (Bruker, Germany) equipped with a CaptiveSpray (Bruker, Germany) ion source. The mass spectra were processed using DataAnalysis 4.1 software to obtain the mass lists, following a preset script to analyze continuous chromatograms. Proteins were identified from the mass lists using the Mascot 2.4.0 program (Matrix Science). Protein representation in the sample was evaluated using Multi Quant 3.0.2 software from the MRM transition peak area.

### *2.6. Obtaining a Line of Human Juvenile Chondroblasts*

We used a culture of juvenile chondroblasts obtained from cartilage fragments of the interphalangeal joints of removed extra toes in healthy children diagnosed with polydactyly. Collection of biological material for cell culture was performed after parents/legal representatives had signed voluntary informed consent and with approval from the Bioethics Committee at Samara State Medical University. Tissue donors were somatically healthy and negative for HPV, HIV, HBsAg, and HCV infections. Material preparation was performed in the Biotech Department "BioTech" cell culture laboratory at Samara State Medical Univer-

sity. This laboratory is equipped with a suite of class B "clean rooms", with the possibility of upgrading to class A areas following ISO 5 standard. Cartilage tissue fragments were washed three times with sterile Hanks' solution, mechanically crushed, and then subjected to enzymatic treatment with 0.1% collagenase solution Biolot LLC (Saint Petersburg, Russia) for two hours in a shaker incubator BioSan (Riga, Latvia). The enzyme was inactivated by adding sterile 0.02% Versene solution (Biolot LLC, Russia). The material was transferred to centrifuge tubes and was washed in complete growth medium 199 containing 10% fetal calf serum (Biolot LLC, Russia) and spun at 1500 rpm on a low-temperature centrifuge Eppendorf 572 R (Eppendorf SE, Hamburg, Germany) at 4 ◦C for 20 min. The pellet was transferred into sterile plastic culture dishes (TRR Techno Plastic Products AG, Trasadingen, Switzerland) with an area of 25 cm2. A fresh portion of 10% 199 complete growth medium was added to the culture vial, then placed in a CO2 incubator. The condition of cells in the culture vial was monitored daily by observation under an inverted microscope Olympus CKX-41 (Olympus Corporation, Tokyo, Japan). The culture medium was changed every three days until the culture had attained 80% confluence, at which point the cells were transferred into new culture vials. The obtained chondroblast lines were analyzed for viability at the second passage using fluorescence microscopy.

#### *2.7. Creation of a Cell-Tissue Graft*

The Lyoplast® tissue engineering product is a 3D carrier of demineralized human spongiosa seeded with juvenile chondroblasts. The chondroblasts were seeded on the scaffold. Cell culture was removed from the bottom of the culture plate at four passages in a traditional manner and seeded at a density of 5 × 104 cells per 27 mm<sup>3</sup> (3 mm × 3 mm × 3 mm) block of the medium. The tissue-engineered constructs were placed in 7 mL vials with a complete growth medium (2 samples per vial) and were cultured in an incubator at 5% CO2 and 20% O2 at 37 ◦C. Samples of demineralized spongiosa Lyoplast® of similar size but without chondroblasts served as a control group.

#### *2.8. Fluorescence Microscopy*

Fluorescence microscopy was performed using the Leica DMIL LED fluorescence module (Germany). For this purpose, a Live/Dead® Viability/Cytotoxicity AssayKitfluorophore kit (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used. Staining was performed according to the manufacturer's protocol. The kit contains Calcein-AM and ethidium homodimer-1 solutions and is designed for simultaneous fluorescent staining of live and dead cells. Calcein-AM, the acetoxymethyl ester of calcein, is highly lipophilic and permeable to cell membranes. Although calcein-AM is not a fluorescent molecule, calcein produced by esterase reaction with calcein-AM in viable cells emits strong green fluorescence (490 nm excitation, 515 nm emission), while calcein-AM stains only viable cells. On the other hand, the nucleus staining dye ethidium homodimer-1 cannot pass through cell membranes, but it penetrates the membrane of dead cells, reaches the nucleus, and intercalates with the double helix of the cell DNA, where it shows red fluorescence (535 nm excitation, 617 nm emission). Thus, living cells glow green, while dead cells glow red.

#### *2.9. MTT Assay for the Study of Demineralized Human Spongiosa Cytotoxicity*

A culture of juvenile human chondroblasts of the seventh passage was used to study the cytotoxicity of the human spongiosa. A culture of human chondroblasts was seeded on a 24-well plate (SPL LIFE SCIENCES, Gyeonggi-do, Korea) at a dose of 4 × <sup>10</sup><sup>4</sup> cells/well and incubated in a CO2 incubator Sanyo MSO-18AC (SANYO Electric Co., Ltd., Osaka, Japan) at 37 ◦C and 5% CO2. Control №1—medium only; control №2—medium with spongiosa. Probes №1 and №2 are cells only and cells with spongiosa, respectively. After the monolayer reached 80% confluency, the nutrient medium 199 Biolo t LLC, (Saint Petersburg, Russia) with a serum content of 10% (Biolo T, Russia) was changed in the wells, and Lyoplast® spongiosa samples Lyocell (Samara, Russia) were placed in cubes 5 mm × 5 mm × 5 mm in size. Permeable plate inserts SPL LIFE SCIENCES, Gyeonggi-do,

Korea were used to hold the material above the monolayer to exclude mechanical damage to the monolayer. After 48 h of cultivation with the material, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), which allows evaluating the ability of live cells to convert soluble tetrazolium salt into an insoluble purple precipitate of formazan due to the action of cellular dehydrogenases. After removing the medium, each well was incubated with 0.3 mg/mL MTT in a growth medium at 37 ◦C for 3.5 h. At the end of the incubation period, the medium was removed by pipetting; intracellular formazan was dissolved in 200 μL of DMSO; and the optical density was measured at 550 nm on a Tecan Infinite M200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The percentage of viable cells was determined based on the calculated optical density, taking as 100% the values in the wells with cells without material.
