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.
This is a preview of subscription content, log in to check access.
This study was supported by project 15-25813A awarded by the Ministry of Health of the Czech Republic and PROGRES Q26 provided by Charles University, Czech Republic. Special thanks go to Blanka Bilkova for her technical assistance.
Compliance with ethical standards
Conflict of interest
The authors declare no conflicts of interest.
All experiments were carried out according to the guidelines for the care and use of experimental animals and approved by the Resort Professional Commission of the Czech Academy of Sciences for Approval of Projects of Experiments on Animals (Approved protocol No 32/2015 and 53/2015).
Deligianni DD, Katsala ND, Koutsoukos PG, Missirlis YF (2001) Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials 22:87–96CrossRefPubMedGoogle Scholar
Diederichs S, Röker S, Marten D et al (2009) Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z® RP Platform. Biotechnol Prog 25:1762–1771PubMedGoogle Scholar
Gerecht-Nir S, Cohen S, Itskovitz-Eldor J (2004) Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation. Biotechnol Bioeng 86:493–502CrossRefPubMedGoogle Scholar
Hempel U, Preissler C, Vogel S et al (2014) Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts. Biomed Res Int 2014:e938368. https://doi.org/10.1155/2014/938368CrossRefGoogle Scholar
Kuboki Y, Takita H, Kobayashi D et al (1998) BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res 39:190–199CrossRefPubMedGoogle Scholar
O’Brien FJ, Harley BA, Waller MA et al (2007) The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering. Technol Health Care 15:3–17CrossRefGoogle Scholar
Prosecká E, Rampichová M, Litvinec A et al (2015) Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte-rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo. J Biomed Mater Res A 103:671–682CrossRefPubMedGoogle Scholar
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676CrossRefGoogle Scholar
Schumacher M, Uhl F, Detsch R et al (2010) Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique. J Mater Sci Mater Med 21:3039–3048CrossRefPubMedGoogle Scholar
Shuai C, Li Y, Wang G et al (2019) Surface modification of nanodiamond: toward the dispersion of reinforced phase in poly-l-lactic acid scaffolds. Int J Biol Macromol 126:1116–1124CrossRefPubMedGoogle Scholar
Varley MC, Neelakantan S, Clyne TW et al (2016) Cell structure, stiffness and permeability of freeze-dried collagen scaffolds in dry and hydrated states. Acta Biomater 33:166–175CrossRefPubMedGoogle Scholar
Villa MM, Wang L, Huang J et al (2015) Bone tissue engineering with a collagen–hydroxyapatite scaffold and culture expanded bone marrow stromal cells. J Biomed Mater Res B Appl Biomater 103:243–253CrossRefPubMedGoogle Scholar
Wang Y, Tomlins PE, Coombes AG, Rides M (2009) On the determination of Darcy permeability coefficients for a microporous tissue scaffold. Tissue Eng Part C Methods 16:281–289CrossRefGoogle Scholar
Wen D, Androjna C, Vasanji A et al (2010) Lipids and collagen matrix restrict the hydraulic permeability within the porous compartment of adult cortical bone. Ann Biomed Eng 38:558–569CrossRefPubMedGoogle Scholar
Yavari SA, Wauthlé R, van der Stok J et al (2013) Fatigue behavior of porous biomaterials manufactured using selective laser melting. Mater Sci Eng C 33:4849–4858CrossRefGoogle Scholar