In vitro characterization of hierarchical 3D scaffolds produced by combining additive manufacturing and thermally induced phase separation

Abstract

This paper reports on the hybrid process we have used for producing hierarchical scaffolds made of poly(lactic-co-glycolic) acid (PLGA) and nanohydroxyapatite (nHA), analyzes their internal structures via scanning electron microscopy, and presents the results of our in vitro proliferation of MC3T3-E1 cells and alkaline phosphatase activity (ALP) for 0 and 21 days. These scaffolds were produced by combining additive manufacturing (AM) and thermally induced phase separation (TIPS) techniques. Slow cooling at a rate of 1.5 °C/min during the TIPS process was used to enable a uniform temperature throughout the scaffolds, and therefore, a relatively uniform pore size range. We produced ten different scaffold compositions and topologies in this study. These scaffolds had macrochannels with diameters of ∼300 µm, ∼380 µm, and ∼460 µm, generated by the extraction of embedded porous 3D-plotted polyethylene glycol (PEG) matrices. The other experimental factors included different TIPS temperatures (−20 °C, −10 °C, and 0 °C), as well as varying PLGA concentrations (8%, 10%, and 12% w/v) and nHA content (0%, 10%, and 20% w/w). Our results indicated that almost all these macro/microporous scaffolds supported cell growth over the period of 21 days. Nevertheless, significant differences were observed among some scaffolds in terms of their support of cell proliferation and differentiation. This paper presents the results of our in vitro cell culture for 0 and 21 days. Our optimal scaffold with a porosity of ∼90%, a modulus of ∼5.2 MPa, and a nHA content of 20% showed a cell adhesion of ∼29% on day 0 and maintained cell proliferation and ALP activity over the 21-day in vitro culture. Hence, the use of additive manufacturing and designed experiments to optimize the scaffold fabrication parameters resulted in superior mechanical properties that most other studies using TIPS.

Keywords:

• Cell differentiation
• cell proliferation
• scaffolds
• tissue engineering
• phase separated polymer
• 3D-plotting

Acknowledgements

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. In addition, we acknowledge the funds from the Ohio Board of Regents and the Ohio Third Frontier Program. The authors also thank the technical contributions of Songmi Koo, Junyi Liu, Riley Sheppard, Joshua Silverstein, Matthew Duley, Jackson Conroy, Ben Marks, and Douglas Hart, as well as the administrative assistance of Laurie Edwards.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Research reported in this paper was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 1R15AR066269-01A1 (Yousefi–James).