*Article* **Biopolymer Material from Human Spongiosa for Regenerative Medicine Application**

**Ilya L. Tsiklin , Evgeniy I. Pugachev, Alexandr V. Kolsanov, Elena V. Timchenko \*, Violetta V. Boltovskaya, Pavel E. Timchenko and Larisa T. Volova**

> Biotechnology Center "Biotech", Samara State Medical University, 443079 Samara, Russia; tsiklin.i@yandex.ru (I.L.T.); e.i.pugachev@samsmu.ru (E.I.P.); avkolsanov@mail.ru (A.V.K.); violetta.boltovskaya@yandex.ru (V.V.B.); timpavel@mail.ru (P.E.T.); volovalt@yandex.ru (L.T.V.) **\*** Correspondence: laser-optics.timchenko@mail.ru; Tel.: +79-277-111-387

**Abstract:** Natural biopolymers demonstrate significant bone and connective tissue-engineering application efficiency. However, the quality of the biopolymer directly depends on microstructure and biochemical properties. This study aims to investigate the biocompatibility and microstructural properties of demineralized human spongiosa Lyoplast® (Samara, Russian Federation). The graft's microstructural and biochemical properties were analyzed by scanning electron microscopy (SEM), micro-computed tomography, Raman spectroscopy, and proteomic analysis. Furthermore, the cell adhesion property of the graft was evaluated using cell cultures and fluorescence microscopy. Microstructural analysis revealed the hierarchical porous structure of the graft with complete removal of the cellular debris and bone marrow components. Moreover, the proteomic analysis confirmed the preservation of collagen and extracellular proteins, stimulating and inhibiting cell adhesion, proliferation, and differentiation. We revealed the adhesion of chondroblast cell cultures in vitro without any evidence of cytotoxicity. According to the study results, demineralized human spongiosa Lyoplast® can be effectively used as the bioactive scaffold for articular hyaline cartilage tissue engineering.

**Keywords:** biopolymers; demineralized human spongiosa; scanning electron microscopy; micro-computed tomography; Raman spectroscopy; proteomic analysis; chondroblasts; tissue engineering; scaffold; fluorophores
