Page 44 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

Calcium deposition and ALP activity by MC3T3-E1 pre-osteoblasts on surface-modified and unmodified PCL scaffolds
Optical images of the alizarin red stained cell/scaffold construct showed cellular calcium deposition (Fig. 7a). The red stain represents areas with calcium minerals. More red stain was observed on 24 h NaOH-treated scaffolds indicating more calcium deposition on this scaffold compared to other scaffolds tested. Scarce calcium deposition was observed on unmodified controls, 72 h NaOH-treated scaffolds, and RGD-immobilized scaffolds. Optical density of the extracted stain from 24 h NaOH-treated scaffolds was increased by 1.9-fold compared to unmodified controls (p<0.001; Fig. 7b). This quantitatively verified the higher levels of calcium deposition observed in figure 7a. No significant difference in calcium deposition was observed between unmodified control, 72 h NaOH-treated, and RGD-immobilized scaffolds. ALP activity of MC3T3-E1 pre-osteoblasts on NaOH-treated, RGD-immobilized, and unmodified PCL scaffolds was measured at day 14 and normalized to total protein content (Fig. 7c). ALP activity on the 24 h NaOH-treated scaffolds was increased by 4.9-fold compared to unmodified controls (p<0.0001). Moreover, ALP activity on the RGD-immobilized scaffolds was increased by 2.0-fold compared to unmodified controls (p<0.01). NaOH treatment for 72 h did not increase the ALP activity compared to unmodified controls.
DISCUSSION
Surface modification of tissue engineering scaffolds plays a key role in improving the performance of scaffolds since cell-scaffold interactions are strongly influenced by the physicochemical properties of the scaffold surface [35]. PCL is widely used for fabrication of bone tissue engineering scaffolds, but it does not support cell attachment and/or proliferation due to its surface hydrophobicity and inertness [36]. Chemical surface modification of PCL with NaOH to introduce functional carboxyl and hydroxyl groups has received much attention [16,23,37,38]. Immobilization of RGD peptide on the surface of PCL has also been used to facilitate cell attachment and subsequent proliferation [19,20]. The cellular response to the same surface modification may be different when the scaffold is a film, a porous spongy structure, or when the scaffold consists of 3D-printed strands. This study aimed to test the proliferation and osteogenic differentiation potential of chemically surface-modified versus RGD-immobilized 3D-printed PCL scaffolds containing MC3T3-E1 pre-osteoblasts. We found that (i) NaOH-treated scaffolds, but not RGD-immobilized scaffolds, displayed a honeycomb-like surface pattern; (ii) seeding
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