Bioprinting of Alginate-Encapsulated Pre-osteoblasts in PLGA/β-TCP Scaffolds Enhances Cell Retention but Impairs Osteogenic Differentiation Compared to Cell Seeding after 3D-Printing

Yasaman Zamani, Javad Mohammadi*, Ghassem Amoabediny, Marco N. Helder, Behrouz Zandieh-Doulabi, Jenneke Klein-Nulend

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Background: In tissue engineering, cellularization of scaffolds has typically been performed by seeding the cells after scaffold fabrication. 3D-printing technology now allows bioprinting of cells encapsulated in a hydrogel simultaneously with the scaffold material. Here, we aimed to investigate whether bioprinting or cell seeding post-printing is more effective in enhancing responses of pre-osteoblastic MC3T3-E1 cell line derived from mouse calvaria. Methods: Two methods were used for incorporation of pre-osteoblasts in poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds: first, seeding the cells after scaffold printing, and second, bioprinting of alginate-encapsulated cells between PLGA/β-TCP struts. AlamarBlue® assay was used to determine cell retention and proliferation. Live/dead staining and picrosirius red staining were used to assess cell viability and collagen deposition, respectively. Alkaline phosphatase activity was measured to assess the osteoblastic phenotype of pre-osteoblasts. Results: Cell retention was 51 ± 5% in cell-seeded and 87 ± 3% in bioprinted scaffolds, whereas cell viabilities were 87 ± 2% and 78 ± 4%, respectively. Cell proliferation was higher on cell-seeded scaffolds (day 7: 2.5-fold, day 14: 4.0-fold, day 21: 4.7-fold) compared with bioprinted scaffolds (1.2-fold, 2.1-fold, and 3.9-fold, respectively). At day 21, the voids between PLGA/β-TCP struts were covered with cell-deposited collagenous matrix in both scaffold types. Alkaline phosphatase activity was higher (3.4-fold) on cell-seeded scaffolds compared with bioprinted scaffolds. Conclusion: Our data demonstrate that encapsulation of pre-osteoblasts in alginate and printing inside PLGA/β-TCP scaffolds enhances cell retention but decreases cell proliferation and osteogenic differentiation compared to seeding cells on scaffolds post-printing. This might have important implications for bone tissue engineering. Lay Summary: Bioprinting, i.e., printing of hydrogel-encapsulated cells alone or in combination with scaffold material, presents an attractive tool for fabrication of customized tissue equivalents as opposed to conventional cell seeding on tissue engineering scaffolds. However, the function of the printed cells might be adversely affected by the hydrogel encapsulation and printing process. Our aim was to investigate whether bioprinting of alginate-encapsulated pre-osteoblasts in poly(lactic-co-glycolic) acid/β-tricalcium phosphate scaffold is more effective in enhancing cell retention, proliferation, and osteogenic differentiation as opposed to cell seeding post-printing. Our studies showed that the bioprinted scaffold had enhanced cell retention capacity, but impaired cell proliferation and osteogenic differentiation compared to the cell-seeded scaffold. These findings might have important implications for the improvement of alginate-based bioinks with, e.g., (natural) bioactive peptides for bioprinting of bone tissue engineering scaffolds. [Figure not available: see fulltext.].

Original languageEnglish
JournalRegenerative Engineering and Translational Medicine
DOIs
Publication statusAccepted/In press - 1 Jan 2020

Cite this