• Arrayed Hollow Channels for Enhanced Oxygen Transport in Three Dimensional-porous Silk Scaffolds Stimulate Osteogenic Activity of Pre-osteoblasts for Bone Tissue Engineering
  • Hadi Seddiqi,1 Alireza Saatchi,2 Ghassem Amoabediny,3 Jianfeng Jin,4 Behrouz Zandieh-Doulabi,5 Jenneke Klein-Nulend,6,*
    1. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences
    2. School of Chemical Engineering, Faculty of Engineering, University of Tehran
    3. School of Chemical Engineering, Faculty of Engineering, University of Tehran
    4. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences
    5. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences
    6. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences


  • Introduction: Cell-seeded scaffolds play a key role in bone tissue formation during bone regeneration. Silk fibroin is a promising natural biopolymer to promote effective bone regeneration, because of its versatile processability, controlled degradability, biocompatibility, hydrophilicity, and compression resistance, as well as its ability to stimulate cell adhesion, proliferation, and differentiation. A major obstacle for clinical application of 3D-porous silk fibroin scaffolds for bone regeneration is a high death rate of cells throughout the scaffold under low oxygen condition. Cell growth and distribution can be accelerated inside a 3D-prous silk fibroin scaffold by using hollow channels, since these channels promote oxygen and nutrient delivery to the central zone of the scaffold. More detailed knowledge of oxygen diffusion, as well as cell proliferation and distribution inside channeled 3D-porous scaffolds is still needed to further promote bone cell behavior inside 3D-porous scaffolds. Therefore, in this study we aimed to investigate whether arrayed hollow channels would further improve oxygen diffusion and enhance cell behavior, i.e. pre-osteoblast viability, proliferation, penetration depth, differentiation, and mineralization inside 3D-porous silk fibroin scaffolds by using finite element (FE) modeling and experiments.
  • Methods: Scaffold fabrication: 3D-porous silk fibroin scaffolds without and with channels of 0.5 or 1 mm diameter were fabricated (scaffold diameter: 2 cm; scaffold height: 1 cm; channel number: 12; channel diameter: 0.5, and 1 mm). Scaffold characterization: Physicomechanical properties of 3D-porous silk fibroin scaffolds, i.e. pore structure (scanning electron microscopy (SEM)), pore size distribution (image J software), and water uptake, as well as mechanical properties, i.e. compression modulus, and ultimate compression strength (universal compression testing machine) were determined. Finite element (FE) modeling: FE modeling was used to quantify the oxygen and cell density distribution inside 3D-porous silk fibroin scaffolds without or with channels of 0.5 and 1 mm diameter during 14 days. Cell culture and scaffold bioactivity: MC3T3-E1 pre-osteoblasts were seeded at 5ˣ105 cells/cm3 on 3D-porous silk fibroin scaffolds, and cultured up to 21 days. Oxygen distribution (Oxygen Sensor Microx TX3 PreScens), as well as pre-osteoblast spreading (SEM), proliferation (AlamarBlue® fluorescent assay), and expression of proliferation and osteogenic genes (RT-PCR) were determined. Statistical analysis: Data are mean±SD from at least 3 independent, separate experiments. Differences were tested with two-way ANOVA combined with Tukey’s multiple comparison test using GraphPad Prism® 8.0, and considered significant if p<0.05.
  • Results: FE modeling: FE modeling revealed a significantly more uniform and homogeneous oxygen concentration, as well as higher cell proliferation inside 1 mm channeled 3D-porous scaffolds than inside non-channeled and 0.5 mm channeled scaffolds. Physicomechanical properties: Experimental results indicated that the scaffold contained a network of interconnected pores with a diameter ranging from 50 to 200 µm, with an average pore diameter of 66.6 ± 24.9 µm (mean ± SD). Normalized water uptake during 2 h was higher inside 1 mm channeled scaffold than in non-channeled and 0.5 mm channeled scaffolds. Compressive modulus was not significantly different between the non-channeled, 0.5 and 1 mm channeled scaffolds. Oxygen transport: the oxygen concentration significantly increased inside 0.5 mm and 1 mm channeled scaffolds (2.3-2.4-fold) compared to non-channeled scaffolds at day 9. Cell proliferation: the cell number was higher inside 0.5 mm and 1 mm channeled scaffolds (1.1-1.3-fold) compared to non-channeled scaffolds at day 14. Gene expression: Expression levels of proliferation marker gene Ki67, and osteogenesis-related genes Runx2, Ocn, Fgf2, Dmp1, and Mepe were assessed after 4, 7, 14, and 21 days. Fgf2 expression was significantly enhanced inside 0.5 mm and 1 mmm channeled scaffolds after 4 days, while decreased after 21 days. Ki67 and Runx2 expression was significantly enhanced inside 0.5 mm channeled scaffolds after 7 days. Moreover, Ocn expression was significantly enhanced inside 0.5 mm channeled scaffolds after 21 days. In addition, Mepe expression was significantly decreased inside 0.5 mm and 1 mm channeled scaffolds after 14 days.
  • Conclusion: In conclusion, arrayed hollow channels inside 3D-silk fibroin scaffolds successfully improved oxygen transport, cell viability, spreading, and proliferation, as well as osteogenic activity of the scaffolds. The mechanical properties of the channeled scaffolds were not significantly different from non-channeled scaffolds. These results are promising to further develop innovative 3D scaffolds containing arrayed hollow channels for bone tissue engineering.
  • Keywords: Bone tissue engineering Channeled 3D-silk scaffold FE modeling Oxygen delivery Pre-osteoblast