Using Microdevice Arrays to Identify 3D Mechanical Stimulation Conditions for Tissue Engineering

Liu, Haijiao 1, 2; Usprech, Jenna 2; Sun, Yu 1, 2; Simmons, Craig 1, 2

1. Department of Mechanical and Industrial Engineering, University of Toronto; 2. Institute of Biomaterials and Biomedical Engineering, University of Toronto

3D mechanical stimulation of cell-seeded biomaterials is important in connective and cardiovascular tissue engineering and is commonly used to induce matrix synthesis in vitro for desired mechanical properties. However, current platforms for 3D mechanical stimulation of engineered load-bearing tissues have limited throughput and capability to investigate optimal mechanical loading regimes (e.g., strain magnitude and duty cycle). State-of-the-art selection of mechanical conditioning protocols has been based on best guesses. Here we demonstrate, for the first time, the use of our deformable membrane device arrays to screen and identify beneficial 3D mechanical stimulation conditions for engineered cell-hydrogel constructs.

Human mesenchymal stromal cell-seeded polyethylene glycol norbornene hydrogel arrays were bound on off-stoichiometry thiol-ene based polydimethylsiloxane membranes, cultured with media supplemented with ascorbic acid (100 µM), and stimulated at 0.1 Hz for all conditions for a week. A set of combinations of 3D mechanical loading conditions was generated via factorial design to examine for their effects on cellular functions and induction of matrix production in vitro. Strain magnitude (5%, 8.5% and 12% nominal tensile strain) and duty cycle (3hrs, 6hrs and 9hrs ON/OFF) were selected based on their reported roles as potent regulators of tissue growth. Commercial pressure regulators with in-house electronics were employed to regulate pressure and actuate multiple devices in parallel. Cell responses were measured by co-staining a-smooth muscle actin (SMA), as a marker of myofibroblast differentiation, and collagen type I for collagen production.

After a week of culture, there was minimal cell spreading in the static control groups based on SMA staining. In comparison, cells in conditions ‘5%, 3hrs’ and ‘8.5%, 6hrs’ had significantly lower sphericity values, indicating a higher extent of cell spreading. In all conditions, cells produced collagen by day 7, as measured by the normalized volume of collagen per cell. However, the ‘5%, 3hrs’ and ‘8.5%, 6hrs’ protocols demonstrated significantly more spread cells with strong collagen staining at the end of the filopodia-like protrusions, indicating an advanced stage of collagen secretion. Interestingly, switching the duty cycle from 3hrs to a less frequent 9hrs period with 5% tensile strain significantly decreased the number of cells with strong collagen staining. Similarly, increasing the strain level from 5% to 12% with 3hrs duty cycle also decreased the portion of cells with strong collagen staining, implying a significant interaction between strain magnitude and duty cycle in regulating cell function.

On-going work is focused on completing the design with complemental conditions to gain a more comprehensive understanding of the relative significance of strain magnitude, duty cycle, and interactions on cell responses. Identified conditions will be further validated with long-term culture