One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

Biomedical Centre Faculty of Medicine in Pilsen Charles University Alej Svobody 1655 76 Pilsen 323 00 Czech Republic

Department of Composites and Carbon Materials Institute of Rock Structure and Mechanics Academy of Sciences of the Czech Republic 5 Holesovickach 41 Prague 182 09 Czech Republic

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University Karlovarská 48 Plzeň 301 00 Czech Republic

Dipartimento di Elettronica Informazione e Bioingegneria Politecnico di Milano Piazza Leonardo da Vinci 32 201 33 Milan Italy

Institute of Dental Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Katerinska 32 Prague 121 08 Czech Republic

Institute of Pathological Physiology 1st Faculty of Medicine Charles University U nemocnice 5 Prague 128 53 Czech Republic

Laboratory of Biomechanics Department of Mechanics Biomechanics and Mechatronics Faculty of Mechanical Engineering Czech Technical University Prague Technicka 4 Prague 166 07 Czech Republic

Laboratory of Cell Regeneration and Plasticity Institute of Animal Physiology and Genetics Academy of Sciences of the Czech Republic Rumburska 89 Libechov 277 21 Czech Republic

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