Developing a Microloading Platform for Applications in Mechanotransduction Research Karan S Shah1, Spencer L York1, Palaniappan Sethu2, Marnie M Saunders1

1Department of Biomedical Engineering, The University of Akron, Akron, OH 44325 2Center for Biomedical Engineering, University of Louisville, Louisville, KY 40208

Introduction: • Polydimethylsiloxane (PDMS) is an elastic polymer that is used as a cell substrate in

Microsystems applications because of its many diverse properties, including optical clarity, low-cost, non-toxicity, and fabrication ease [1].

• Bone mechanotransduction is the area of research focusing on elucidating the pathways and mechanisms by which bone cells sense and respond to a mechanical load.

• Mechanotransduction models enable investigators to stimulate cells in a physiologically relevant and accurate manner. A variety of cell stimulation devices have been developed over the years and have been classified as to the type of mechanical loading the system imparts [2].

• Here, the objective was to develop a loading platform that would enable a complex, 3D load to be applied to osteocytes in a well-characterized model.

Determination of PDMS Material Properties: • Thin PDMS membranes of 100-2000 μm thick were fabricated via a spin casting procedure.

Briefly, PDMS (Sylgard 182, Dow Corning, Midland, MI) pre-polymer and cross-linking agent were mixed thoroughly in a 10:1 ratio and degassed in a vacuum desiccator to remove trapped air bubbles.

• N=9 PDMS strips of 6.35 mm x 25.4 mm strips were loaded onto a mechanical loading platform outfitted with a 44.48 N load cell and 25 mm displacement sensor.

• Strips were tested to failure at a rate of 0.3 mm/s with load-displacement recorded in real-time. • For non-contact strain analysis the testing gage length was divided into three equal regions

(top, middle, lower) with India ink and a video camera setup was used to record the tensile test.

Parametric Modeling of Substrate Geometry: • A three-dimensional computer model of the PDMS substrate was built using solid modeling

software (SolidWorks). • A well diameter of 7 mm was conveniently selected to enable future studies to utilize a native

bone matrix background and was the one dimension held constant in the FEA. The thickness of the well base and the loading platen were parametrically analyzed.

• Three well membrane thicknesses were evaluated: 0.25 mm, 0.38 mm, and 0.5 mm. Three platen diameters were evaluated (2 mm, 4 mm, and 6 mm).

• For a given thickness and platen diameter, a load controlled test was simulated with loads of 0.25 N, 0.50 N and 0.75 N. Low load values were selected to make sure the PDMS substrate did not overtly exceed the yield stress of 2.59 MPa and the maximum displacements and strains remained in the desirable micron range.

Microloading Platform Development: • The micro-actuated loading device consisted of an aluminum base plate connected to a

Plexiglas® top plate. • A smaller aluminum plate (75 mm X 75mm X 10 mm) was suspended from the Plexiglas® top

plate. • The second tier served as a mounting plate for the actuator (Zaber Technologies, T-NA08A25). • As shown in Figures 5 and 6, the actuator connects to a load cell (2.452 N) that in turn connects

to a platen. • A square cutout on the Plexiglas® plate allows the platen to extend beyond the mounting plate

and make contact with the PDMS well substrate.

Discussion: • Stress-strain curves for the PDMS strips are shown in Figure 1. For an average thickness of

0.38 mm, the samples yielded a consistent average Young’s modulus of 14.47 MPa and an average ultimate tensile strength (UTS) of 2.59 MPa.

• The results of the fabricated PDMS samples from the tensile test are comparable with the generally acceptable values [4].

• Because the intended use of the PDMS was as a substrate for microloading bone cells, it was necessary to characterize the strain behavior of the material well below the 25% strain range. Results showed that this strain range resulted in uniform, linear loading in all three regions (top, middle and lower), Figure 2.

• The finite model results revealed that a membrane thickness of 0.5 mm is required for the use of PDMS in mechanotransduction studies under the platen loading model.

• From the numerical modeling studies, a well membrane thickness of 0.5 mm and a platen diameter of 6 mm were selected to be tested with a microloading platform.

• The microloading platform experiment was performed to confirm the uniformity of the PDMS material at low load ranges. The results, in Figure 7 above, indicated that the experimental data did not correlate well with the FEA models, particularly at higher loading strains.

• The results indicated that additional material characterization, such as relaxation and fatigue, in the microloading range are required.

• Further characterization studies are needed to improve the parametric FE model and achieve a closer correlation between the finite and microloading platform data at lower loads.

Fig. 1 In-house developed small scale loading platform [3]; PDMS tensile test setup; PDMS Stress-Strain Curves obtained

Fig. 2 0 - 25% Strain behavior of PDMS in the 3 regions (top, middle, lower). Shows linearity of material in the given strain range

Fig. 3 FEA study results for 6mm diameter platen. (a) 0.25 mm well thickness with 0.25 N applied load; (b) 0.25 mm well thickness with 0.5 N applied load; (c) 0.25 mm well thickness with 0.75 N applied load; (d) 0.38 mm well thickness with 0.25 N applied load; (e) 0.38 mm well thickness with 0.5 N applied load; (f) 0.38 mm well thickness with 0.75 N applied load; (g) 0.5 mm well thickness with 0.25 N applied load; (h) 0.5 mm well thickness with 0.5 N applied load; (i) 0.5 mm well thickness with 0.75 N applied load Fig. 4 Microloading Loading Platform

Design

Fig. 5 Microloading platform test setup with load cell attached to a platen and a PDMS strip

Fig. 6 Load-Displacement curves obtained from microloading platform test (5 points collected for 6 runs) overlaid with the results from the FEA study for a 0.5 mm well thickness with 6 mm platen model (blue circles)

References: [1] Liu, M., Sun, J., Sun, Y., Bock, C., & Chen, Q. (2009). Thickness-dependent mechanical properties of polydimethylsiloxane membranes. Journal of Micromech. Microeng. 19. [2] Brown, T. D. (2000). Techniques for

mechanical stimulation of cells in vitro: a review. Journal of Biomechanics 33, 3-14. [3] Saunders, M., & Donahue, H. (2004). Development of a cost-effective loading machine for biomechanical evaluation of mouse transgenic models. Med Eng Phys 26, 595-603. [4] Mark, J. (1999). Polymer Data Handbook. New York: Oxford Univ. Press.

Partial support for this work was provided by an NSF CAREER Award and an NIH NIDCR AREA (R15) Award.