Molecular Biology Reports

, Volume 46, Issue 4, pp 4483–4500 | Cite as

Positive impact of dynamic seeding of mesenchymal stem cells on bone-like biodegradable scaffolds with increased content of calcium phosphate nanoparticles

  • Pavla Sauerova
  • Tomas Suchy
  • Monika Supova
  • Martin Bartos
  • Jiri Klima
  • Jana Juhasova
  • Stefan Juhas
  • Tereza Kubikova
  • Zbynek Tonar
  • Radek Sedlacek
  • Marco Piola
  • Gianfranco Beniamino Fiore
  • Monica Soncini
  • Marie Hubalek KalbacovaEmail author
Original Article


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.


Mesenchymal stem cells Collagen scaffolds Dynamic seeding Static cultivation Bone tissue engineering 



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.

Ethical approval

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).


  1. 1.
    Akin FA, Zreiqat H, Jordan S et al (2001) Preparation and analysis of macroporous TiO2 films on Ti surfaces for bone–tissue implants. J Biomed Mater Res 57:588–596.;2-Y CrossRefPubMedGoogle Scholar
  2. 2.
    Alford AI, Kozloff KM, Hankenson KD (2015) Extracellular matrix networks in bone remodeling. Int J Biochem Cell Biol 65:20–31. CrossRefPubMedGoogle Scholar
  3. 3.
    Bourboulia D, Stetler-Stevenson WG (2010) Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion. Semin Cancer Biol 20:161–168. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Camassola M, de Macedo Braga LMG, Chagastelles PC, Nardi NB (2012) Methodology, biology and clinical applications of human mesenchymal stem cells. Methods Mol Biol Clifton NJ 879:491–504. CrossRefGoogle Scholar
  5. 5.
    Caplan AI, Dennis JE (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochem 98:1076–1084. CrossRefPubMedGoogle Scholar
  6. 6.
    Carreira AC, Alves GG, Zambuzzi WF et al (2014) Bone morphogenetic proteins: structure, biological function and therapeutic applications. Arch Biochem Biophys 561:64–73. CrossRefPubMedGoogle Scholar
  7. 7.
    Chaturvedi LS, Marsh HM, Basson MD (2007) Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells. Am J Physiol 292:C1701–C1713. CrossRefGoogle Scholar
  8. 8.
    Chen RH, Sarnecki C, Blenis J (1992) Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12:915–927. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cheng Y, Ramos D, Lee P et al (2014) Collagen functionalized bioactive nanofiber matrices for osteogenic differentiation of mesenchymal stem cells: bone tissue engineering. J Biomed Nanotechnol 10:287–298. CrossRefPubMedGoogle Scholar
  10. 10.
    Cooper DML, Matyas JR, Katzenberg MA, Hallgrimsson B (2004) Comparison of microcomputed tomographic and microradiographic measurements of cortical bone porosity. Calcif Tissue Int 74:437–447. CrossRefPubMedGoogle Scholar
  11. 11.
    da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI (2009) Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev 20:419–427. CrossRefGoogle Scholar
  12. 12.
    Dalby MJ, Gadegaard N, Tare R et al (2007) The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater 6:997–1003. CrossRefPubMedGoogle Scholar
  13. 13.
    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
  14. 14.
    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
  15. 15.
    Discher DE, Janmey P, Wang Y (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143. CrossRefPubMedGoogle Scholar
  16. 16.
    Dutta P, Hajra S, Chattoraj DK (1997) Binding of water and solute to protein-mixture and protein-coated alumina. Indian J Biochem Biophys 34:449–460PubMedGoogle Scholar
  17. 17.
    Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689. CrossRefPubMedGoogle Scholar
  18. 18.
    Feng B, Chen J, Zhang X (2002) Interaction of calcium and phosphate in apatite coating on titanium with serum albumin. Biomaterials 23:2499–2507CrossRefPubMedGoogle Scholar
  19. 19.
    Feng P, Wu P, Gao C et al (2018) A multimaterial scaffold with tunable properties: toward bone tissue repair. Adv Sci 5:1700817CrossRefGoogle Scholar
  20. 20.
    Fernekorn U, Hampl J, Augspurger C et al (2013) In vitro cultivation of biopsy derived primary hepatocytes leads to a more metabolic genotype in perfused 3D scaffolds than static 3D cell culture. RSC Adv 3:16558–16568. CrossRefGoogle Scholar
  21. 21.
    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
  22. 22.
    Grier WK, Iyoha EM, Harley BAC (2017) The influence of pore size and stiffness on tenocyte bioactivity and transcriptomic stability in collagen-GAG scaffolds. J Mech Behav Biomed Mater 65:295–305. CrossRefPubMedGoogle Scholar
  23. 23.
    Grimm MJ, Williams JL (1997) Measurements of permeability in human calcaneal trabecular bone. J Biomech 30:743–745CrossRefPubMedGoogle Scholar
  24. 24.
    Gupta D, Venugopal J, Mitra S et al (2009) Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials 30:2085–2094. CrossRefPubMedGoogle Scholar
  25. 25.
    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. CrossRefGoogle Scholar
  26. 26.
    Huang ZL, Liu GY, He Y et al (2012) Interaction between hydroxyapatite and collagen. Adv Mater Res 412:384–387. CrossRefGoogle Scholar
  27. 27.
    Juhásová J, Juhás S, Klíma J et al (2011) Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment. Physiol Res 60:559–571PubMedGoogle Scholar
  28. 28.
    Jungreuthmayer C, Donahue SW, Jaasma MJ et al (2008) A comparative study of shear stresses in collagen-glycosaminoglycan and calcium phosphate scaffolds in bone tissue-engineering bioreactors. Tissue Eng Part A 15:1141–1149. CrossRefGoogle Scholar
  29. 29.
    Karp JM, Dalton PD, Shoichet MS (2003) Scaffolds for tissue engineering. MRS Bull 28:301–306. CrossRefGoogle Scholar
  30. 30.
    Kasten P, Beyen I, Niemeyer P et al (2008) Porosity and pore size of β-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: an in vitro and in vivo study. Acta Biomater 4:1904–1915. CrossRefPubMedGoogle Scholar
  31. 31.
    Keaveny TM, Morgan EF, Niebur GL, Yeh OC (2001) Biomechanics of trabecular bone. Annu Rev Biomed Eng 3:307–333. CrossRefPubMedGoogle Scholar
  32. 32.
    Kemppainen JM, Hollister SJ (2010) Differential effects of designed scaffold permeability on chondrogenesis by chondrocytes and bone marrow stromal cells. Biomaterials 31:279–287. CrossRefPubMedGoogle Scholar
  33. 33.
    Krebs S, Fischaleck M, Blum H (2009) A simple and loss-free method to remove TRIzol contaminations from minute RNA samples. Anal Biochem 387:136–138. CrossRefPubMedGoogle Scholar
  34. 34.
    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
  35. 35.
    Kujala S, Ryhänen J, Danilov A, Tuukkanen J (2003) Effect of porosity on the osteointegration and bone ingrowth of a weight-bearing nickel–titanium bone graft substitute. Biomaterials 24:4691–4697. CrossRefPubMedGoogle Scholar
  36. 36.
    Li W-J, Laurencin CT, Caterson EJ et al (2002) Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J Biomed Mater Res 60:613–621. CrossRefPubMedGoogle Scholar
  37. 37.
    McCoy RJ, Jungreuthmayer C, O’Brien FJ (2012) Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor. Biotechnol Bioeng 109:1583–1594. CrossRefPubMedGoogle Scholar
  38. 38.
    Müller P, Bulnheim U, Diener A et al (2008) Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med 12:281–291CrossRefPubMedGoogle Scholar
  39. 39.
    Nemir S, West JL (2010) Synthetic materials in the study of cell response to substrate rigidity. Ann Biomed Eng 38:2–20. CrossRefPubMedGoogle Scholar
  40. 40.
    Novotna K, Zajdlova M, Suchy T et al (2014) Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells. J Biomed Mater Res A 102:3918–3930. CrossRefPubMedGoogle Scholar
  41. 41.
    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
  42. 42.
    Piola M, Soncini M, Cantini M et al (2013) Design and functional testing of a multichamber perfusion platform for three-dimensional scaffolds. Sci World J. CrossRefGoogle Scholar
  43. 43.
    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
  44. 44.
    Raiskup F, Spoerl E (2013) Corneal crosslinking with riboflavin and ultraviolet A. I. Principles. Ocul Surf 11:65–74. CrossRefPubMedGoogle Scholar
  45. 45.
    Salasznyk RM, Klees RF, Williams WA et al (2007) Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. Exp Cell Res 313:22–37. CrossRefPubMedGoogle Scholar
  46. 46.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676CrossRefGoogle Scholar
  47. 47.
    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
  48. 48.
    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
  49. 49.
    Stops AJF, Heraty KB, Browne M et al (2010) A prediction of cell differentiation and proliferation within a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow. J Biomech 43:618–626. CrossRefPubMedGoogle Scholar
  50. 50.
    Suchỳ T, Šupová M, Bartoš M et al (2018) Dry versus hydrated collagen scaffolds: are dry states representative of hydrated states? J Mater Sci Mater Med 29:20CrossRefPubMedGoogle Scholar
  51. 51.
    Suchý T, Šupová M, Sauerová P et al (2015) The effects of different cross-linking conditions on collagen-based nanocomposite scaffolds—an in vitro evaluation using mesenchymal stem cells. Biomed Mater 10:065008. CrossRefPubMedGoogle Scholar
  52. 52.
    Tian X-F, Heng B-C, Ge Z et al (2008) Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems. Scand J Clin Lab Investig 68:58–67. CrossRefGoogle Scholar
  53. 53.
    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
  54. 54.
    Verdanova M, Sauerova P, Hempel U, Kalbacova MH (2017) Initial cell adhesion of three cell types in the presence and absence of serum proteins. Histochem Cell Biol 148:273–288. CrossRefPubMedGoogle Scholar
  55. 55.
    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
  56. 56.
    Volkmer E, Drosse I, Otto S et al (2008) Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone. Tissue Eng Part A 14:1331–1340. CrossRefPubMedGoogle Scholar
  57. 57.
    Wang S, Liu Y, Fang D, Shi S (2007) The miniature pig: a useful large animal model for dental and orofacial research. Oral Dis 13:530–537. CrossRefPubMedGoogle Scholar
  58. 58.
    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
  59. 59.
    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
  60. 60.
    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
  61. 61.
    Zhang Q, Lu H, Kawazoe N, Chen G (2014) Pore size effect of collagen scaffolds on cartilage regeneration. Acta Biomater 10:2005–2013. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Pavla Sauerova
    • 1
    • 2
  • Tomas Suchy
    • 4
    • 5
  • Monika Supova
    • 4
  • Martin Bartos
    • 1
    • 6
  • Jiri Klima
    • 7
  • Jana Juhasova
    • 7
  • Stefan Juhas
    • 7
  • Tereza Kubikova
    • 2
    • 3
  • Zbynek Tonar
    • 2
    • 3
  • Radek Sedlacek
    • 5
  • Marco Piola
    • 8
  • Gianfranco Beniamino Fiore
    • 8
  • Monica Soncini
    • 8
  • Marie Hubalek Kalbacova
    • 1
    • 2
    Email author
  1. 1.Institute of Pathological Physiology, 1st Faculty of MedicineCharles UniversityPragueCzech Republic
  2. 2.Biomedical Centre, Faculty of Medicine in PilsenCharles UniversityPilsenCzech Republic
  3. 3.Department of Histology and Embryology, Faculty of Medicine in PilsenCharles UniversityPlzeňCzech Republic
  4. 4.Department of Composites and Carbon Materials, Institute of Rock Structure and MechanicsAcademy of Sciences of the Czech RepublicPragueCzech Republic
  5. 5.Laboratory of Biomechanics, Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical EngineeringCzech Technical University in PraguePragueCzech Republic
  6. 6.Institute of Dental Medicine, 1st Faculty of MedicineCharles University and General University Hospital in PraguePragueCzech Republic
  7. 7.Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and GeneticsAcademy of Sciences of the Czech RepublicLibechovCzech Republic
  8. 8.Dipartimento di Elettronica, Informazione e BioingegneriaPolitecnico di MilanoMilanItaly

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