K19 - Dynamically Tunable Substrates to Probe Tendon Pathophysiology

Introduction: : Developing physiologically-mimetic substrates has grown in popularity over recent decades, especially as the FDA recently announced phasing out of the requirement for animal models in preclinical trials. Some substrates, such as magnetorheological elastomers (MREs), while many others are 3-D tissue or organoid models. Realistically, not every research environment has the resources to fabricate or accurately study 3-D models; MREs offer a simplicity that most research environments can easily fabricate and study and have mechanical properties that can mimic the heart, skeletal muscle, and the dermis.

Tendons have elastic moduli of approximately 300 kPa, and pathologies can both increase and decrease moduli, though this is an understudied field. In this study, we develop two novel MRE formulations and manufacturing protocols to study their effects on tendon pathology. MREs are silicone substrates with tunable and reversible mechanical properties in response to a magnetic field. The MREs developed in this abstract have elastic moduli of 300 kPa, with dynamic ranges of ±100 kPa through the addition or subtraction of a magnetic field. Parallel plate rheology confirms that seeded cells will primarily feel the elastic component of the material, through tan(delta) values below 0.2.

Ongoing cell morphology experiments demonstrate active cell remodeling in the minutes and hours after a dynamic stiffening or softening event. To perform an unbiased analysis of the data, a Cell Profiler pipeline was developed to automatically trace the outline of each cell based on the F-actin fluorescence.

Materials and

Methods: : Low-range (LR) and high-range (HR) MREs were fabricated by mixing 527 polydimethylsiloxane (PDMS), 184 PDMS, and carbonyl iron particles (CIPs). 5g of prepolymer was poured into a 35mm dish and cured at 60⁰C for 16.5 and 16 hours, respectively. MRE modulus was altered by exposing the dishes to varied magnetic field strengths through 0.25-inch thick, 1.25-inch diameter N52 disc permanent magnets. Microindentation was performed using a 0.9mm radius probe to determine elastic moduli, while 20mm parallel plate rheology was performed to understand shear and loss moduli. Frequency sweeps were performed at 0.1% strain between 0.2 and 20 rad/s. Strain sweeps confirm 0.1% strain is well within the linear region of the MREs.

ScxGFP tenocytes were isolated from 3-week-old healthy mouse tails. Tails were digested in 2 mg/ml collagenase I at 37 °C for 45 minutes before using a 40 µm nylon mesh for cell filtration. Tenocytes were expanded in hypoxia before the first passage.

Before cell seeding, MREs were sterilized and functionalized with 100μg/mL collagen type I. To probe cell morphology in response to a dynamic change, cells were seeded at 2,000 cells/cm2. After 24 hours of pre-conditioning at a 300 kPa elastic modulus, the elastic modulus was altered in ± 50 or 100 kPa increments, and the tenocytes were fixed with 4% paraformaldehyde at 0.25, 0.5, 1, 2, and 3 hours. Cells were stained with Phalloidin and DAPI and imaged using an upright microscope. A custom CellProfiler pipeline was develpped to automatically extract nuclear and cell phenotypes (Fig. 1).

Results, Conclusions, and Discussions:: Fine tuning of the MRE formulations occurred to achieve LR dynamic modulus of 200-300 kPa, while the dynamic range of the HR modulus was 300-400 kPa. The formulations are shown in Fig. 2A, where 1 part toluene was added for every 5 parts mass of PDMS. Toluene was crucial in reducing prepolymer viscosity and ensuring efficient mixing of CIPs. LR MREs have elastic moduli of 206.9±8.2, 251.9±28.1, and 297.2±21.9 kPa using 0, 1, and 3 0.25-inch-thick magnets, respectively; HR MREs have elastic moduli of 298.9±14.3, 351.5±25.7, and 399.6±28.5 kPa using 0, 2, and 3 magnets, as shown in Fig. 2B.

Parallel plate rheology demonstrates expected, results from Poisson’s ratio: storage modulus should be approximately 1/3 the elastic modulus. The LR MREs demonstrate storage moduli of 58.3±1.9, 80.4±1.2, and 94.0±0.6 kPa for each condition, while HR MREs demonstrate storage moduli of 107.8±2.0, 120.3±0.8, and 132.8±3.2 (Fig. 2C). The LR MREs have tan(delta) values of 0.110±0.009, 0.113±0.017, and 0.155±0.006 for each respective stiffness condition, while HR MREs have values of 0.071±0.010, 0.069±0.008, and 0.085±006 at an angular frequency of 0.2 rad/s. These materials effectively minimize the viscous, or time-dependent, effects, allowing cells to “feel” the elastic modulus, with minimal viscous component influence. The dynamic range of these materials are capable of mimicking various tissue-types, such as skin, the arterial wall, tendon, and cartilage.

Cell phenotype experiments demonstrate time-dependent remodeling of primary mouse tenocytes. While still in progress, preliminary results point towards short-term cytoskeletal remodeling before achieving a new phenotype steady state. Short-term goals of this project include completion of cell morphology quantification and cell genotyping at each timepoint listed. Cell area, aspect ratio, and circularity and nuclear area and aspect ratio will be analyzed to understand how the tenocytes restructure under dynamic loads. Scx, Mmp3, Col1a1, Col3a1, Thbs4, Ctgf, and Sox9 expression will also be analyzed. Longer-term goals include quantification of matrix deposition and ScxGFP expression. Ultimately, the goal of this platform is for wide availability in laboratory environments to probe physiologies and pathologies of relatively stiff tissues, such as tendon and skin.

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