Page 47 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

24 h NaOH-treated scaffolds having strands with sub-micron pores, might be favorable for pre- osteoblast osteogenic differentiation.
We found that surface modification of scaffolds using 3 M NaOH for 24 h resulted in more extracellular matrix deposition compared to modification with 1 or 5 M NaOH for 24 h. This relatively high NaOH concentration (3 M) and long treatment time (24 h) is in contrast to treatment regimes for electrospun and porous PCL scaffolds, which are normally immersed in lower concentrations of NaOH (0.5 or 1 M) or for shorter times (2 or 1 h) to prevent degradation and loss of mechanical integrity of the scaffold [16,23,55,56]. However, in our study using 3D printing, solid PCL strands with no porosity or feature were obtained and therefore, NaOH concentrations lower than 3 M and treatment times shorter than 24 h did not effectively change scaffold hydrophilicity as was evident by semi-spherical shape of water drop on top of the scaffold.
We found that seeding efficiency of both NaOH-treated and RGD-immobilized scaffolds was similar to the seeding efficiency of unmodified PCL indicating that the same number of cells attached to the scaffolds in the first 12 h after cell seeding. This could be explained by the same large 3D architecture of all scaffolds enabling cell entrapment within the structure. Upon continued culturing, cell proliferation was almost the same on all scaffolds after 3 days but diverged at later time points. This suggests that our modifications show their effect on cell proliferation only in later stages and not within 12 h of cell seeding. This is in agreement with published data showing that seeding efficiency is similar on unmodified PCL scaffolds and PCL scaffolds functionalized with hyaluronic acid and β-TCP, while proliferation and osteogenic activity is different in the following days [57]. After 14 days of culture, cell proliferation and collagenous matrix deposition was higher on 24 h NaOH-treated and RGD-immobilized scaffolds compared with unmodified controls, indicating that both 24 h NaOH-treated scaffolds and RGD-immobilized scaffolds are favorable for the enhancement of pre-osteoblast proliferation and matrix deposition.
We found higher ALP activity and calcium deposition, indicating enhanced osteogenic differentiation on 24 h NaOH-treated scaffolds compared to all other groups. Interestingly, the 24 h NaOH-treated scaffolds outperformed the RGD-immobilized counterparts in both osteogenic markers (i.e. ALP activity and calcium deposition), while proliferation was higher on RGD- immobilized compared with 24 h NaOH-treated scaffolds. We speculate that the lower rate of cell proliferation on 24 h NaOH-treated scaffolds compared to RGD-immobilized scaffolds after 7 days reflects an earlier and more efficient induction of osteogenic cell differentiation on 24 h NaOH- treated scaffolds with a concomitant later and decreased osteogenic response on the RGD- immobilized scaffolds. This is in agreement with published data by others showing that cell proliferation and differentiation show a remarkable inverse relationship [58]. This is also consistent
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