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Biomaterials

Biomaterials - Poster Session B

Poster D1 - Genotypic and Phenotypic Analysis of Tenocyte Transitions to Pathophysiological Elastic Moduli

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Every year, 33 million musculoskeletal injuries are reported in the United States, with half of these being tendon-related injuries. Tendonitis is a dynamic process characterized by complex interactions between various cellular and molecular mechanisms, tissue remodeling, inflammation, and structural changes over time. It can manifest in different forms but often results in the softening or stiffening of the tendon extracellular matrix (ECM). Understanding tendonitis is crucial for advancing musculoskeletal health, optimizing athletic performance, improving clinical outcomes, and developing targeted therapies. Current studies often employ substrates with fixed elastic moduli to study tendons in specific pathological states. To build on previous genotypic studies, we developed two magnetorheological elastomer (MRE) formulations. MREs consist of silicone with encapsulated iron such that their mechanical properties are altered in response to a magnetic field. We developed novel MRE formulations to achieve a range of elastic moduli, both physiological (~300kPa) and pathophysiological (~200 and ~400kPa), under various magnetic field strengths. The elastic modulus, which is crucial for tendon mechanical properties and their capacity to withstand tensile loads under pathophysiological conditions, can potentially influence tenocyte genotype and phenotype. Genotypic changes can disrupt the broader tendon environment through ECM organization and composition. Therefore, in this study, we aim to observe genotype and phenotype alterations in tendons as they transition from physiological to pathophysiological elastic moduli. We analyzed genes associated with proliferation, structural integrity, tissue remodeling, and phenotype changes with immunofluorescent staining and imaging.

Materials and

Methods:  Given the broad dynamic range between pathological and physiological tendon conditions, we developed two distinct MRE devices: one for inducing softening and another for inducing stiffening, both from physiological levels. The first MRE formulation consisted of 7% (v/v) Sylgard 184 PDMS, 68% (v/v) Sylgard 527 PDMS, and 25% (v/v) carbonyl iron particles (CIP), and the second was 10%/65%/25%, for the softening and stiffening event, respectively. All PDMS products were prepared according to manufacturer guidelines. We separately prepared 527 and 184 PDMS by weight, combined them with CIP in a 50mL centrifuge tube, and vortexed the mixture for 5 minutes. We poured 5g of the prepolymer MRE mixture into 35mm dishes, degassed, and baked at 60°C overnight.

Disc neodymium (N52) magnets and a custom-built micro-indenter were utilized to determine the elastic moduli for softening and stiffening experiments. For compressive load measurements, a spherical probe was brought into contact with the MRE surface, applying a target load of 20mN at a rate of 50µm/s, and retracted at the same rate. Nine indentations were performed at each magnetic field strength (0 to 350.8mT in 175.4mT increments). Indentation force and displacement were recorded using a capacitance probe and optical linear encoder. From the force indentation curves, the elastic moduli were calculated by fitting the loading portion curves to the Hertz contact force model. Comparisons of elastic moduli data were analyzed using two-way ANOVA with a Tukey post hoc multiple comparisons test to determine if the substrate stiffness was dependent on magnetic field strength.

Results, Conclusions, and Discussions: Our goal was to develop in-vitro dynamic material platforms that simulate the transition of tendons from a physiological to a pathophysiological state, characterized by either a softening or stiffening event. Ultrasound elastography studies typically report the elastic modulus of Achilles tendons to be approximately 300 ± 30 kPa, making this our target for the physiologic condition. Figure 1 shows the dynamic ranges of both the low-range (7%/68%/25%) and high-range (10%/65%/25%) MRE formulations. The low-range and high-range MREs exhibit dynamic ranges of 183.70 ± 16.13kPa to 288.61 ± 14.81kPa and 297.36 ± 10.23kPa to 425.05 ± 7.81kPa, respectively, from the 0mT condition to the 350mT condition. We observed that the physiologic conditions were effectively replicated across the low-range and high-range platforms, with no significant differences detected. Although the literature on the stiffness profile of pathologic tendons is limited, we are confident that both low- and high-range MREs effectively represent pathophysiologic moduli.

We are interested in understanding the genotypic and phenotypic changes of tendon for stiffening and softening events. By preconditioning tenocytes isolated from mice at a physiological elastic modulus before inducing either a stiffening or softening event, we aim to reduce the cellular aspect ratio and increase ECM deposition. The latter will be measured through RT-PCR and de novo synthesis using Cu-free Click Chemistry to label new ECM deposits.

Acknowledgements (Optional):  We thank Dr. Lim for the collaboration idea and providing ScxGFP mouse tenocytes.