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Biomechanics

Biomechanics - Poster Session E

Poster D10 - The Development of an Uniaxial Mechanoculture System for Rapid Elastogenesis

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Elastin is crucial in tissue engineered vascular grafts (TEVGs) due to its primary role as an extracellular matrix (ECM) protein, and it is essential for both mechanical support and biochemical signaling in tissues [1]. Elastin within the vessel wall allows for deformation when pressure is applied, which enables the vessel to maintain its form. A lack of biomechanical compliance in TEVGs is associated with aneurysms and graft failure [2].
Prior studies have utilized several culture techniques to stimulate elastin production. Additives to culture media conditions, such as introducing tropoelastin, ascorbic acid, and copper sulfate can accelerate this process [3,4]. Additionally, biomechanical loading can be applied to cells to trigger an intracellular response that converts the mechanical stimuli into biochemical signals, thereby fostering elastin production [5]. In these cases, cells were cultured onto sheets or tubular constructs made from various biomaterials and mechanically stimulated to produce ECM sheets that have been used in TEVG designs [6].
While these methods can produce mature elastin, the quantities produced are often small, and can significantly vary in time duration depending on the cell type, typically taking up to thirteen weeks [3]. This extended period creates obstacles for generating substantial amounts of elastin within shorter periods. The goal of this study is to design a culture method to rapidly stimulate elastin production using a customized mechanoculture system that applies cyclic axial stretch and to assess this production with non-destructive 2-Photon intravital imaging.

Materials and

Methods:  Biomaterial sheets made from porcine-derived gelatin were solvent-casted using a 10% weight/volume solution of gelatin and hexafluoro-isopropanol (HFP). After residual HFP evaporated from the cast, a 0.5% Genipin solution in 200 proof ethanol was poured over the dish until covered. The dish was covered and placed in a 37℃ on a rocking plate at 50rpm for 24hrs.
Additional sheets were made from polymers including Poly(L-lactide-co-ε-caprolactone) (PLCL), polycaprolactone (PCL), and polyurethane. Each polymer was dissolved in a 10% weight by volume solution and solvent casted. The PCL sheet was electrospun. All sheets were cut into 2.5 mm x 2.5 mm strips and cultured in a CO2 incubator at 37℃.
For this study, rat fetal lung fibroblast (RFL-6) cells, explanted rat aorta, and heart tissues were used. RFL-6 cells were cultured in flasks for approximately 4 months before being scraped onto sheets. Explanted tissues were cut and distributed across sheets. RFL-6 cells were cultured in F-12K Media with 20% fetal bovine serum and 1% Pen Strep and Amphotericin B (Gibco), while the rat explants were cultured in in Rat Smooth Muscle Cell Media (Cell Applications, USA).
To initiate uniaxial mechanical stretching, a custom designed mechanoculture system (CellScale, Ontario, CA) was employed. This device allows sheets to be clamped and stretched while in culture, with accompanying software to control and monitor stretching conditions. Intravital images were acquired using a 2-Photon microscope before, during, and after mechanical stretching to observe Second Harmonic Generation (SHG) signals and elastin specific autofluorescence (2PEF).

Results, Conclusions, and Discussions: Results
We validated that the system hardware could be sterilized by physical debridement, withstand exposure to bleach, enzymatic soaps, ethanol, and can be autoclaved. Cellularized sheets withstand physical loading into the system without breaking sterility. When sheets were inserted with media, the system was able to perform repeated cyclic stretches with varying magnitudes using the accompanying CellScale software.
After testing various sheets and materials, we devised how to load and unload sheets containing cells without significant damage to testing samples. We determined the maximum stretching throw of the system to be 7 mm. After testing various options, we found that 0.2 mm thick PLCL sheets were the ideal substrate for the mechanoculture device based on physical handleability. PLCL sheets achieved a 246% strain until failure, allowing for a larger range of mechanical testing in future studies. It was determined that pre-treatment of the sheets in 70% ethanol, followed by sterile phosphate buffer solution rinses, and UV light exposure was sufficient for sterilization. RFL-6 cells were found to grow onto the surface of the PLCL without infiltrating the sheet, assisting with our ability to image the samples microscopically.
Discussion
Our customized mechanoculture system was demonstrated as a suitable device for in vitro elastogenesis studies. A critical aspect of this work is the compatibility of this mechanoculture with non-destructive intravital imaging using a 2-Photon microscope. The results in this study illustrate a proof of concept for a mechanical-culturing device and device-loading compatible biomaterial substrates that allow for cell growth and mechanical stimulus as the starting point to investigate what drives soft tissue elastogenesis in vitro.
Conclusion
This study has produced the groundwork for rapid elastin deposition due to mechanical stretching. Multiple sheets were evaluated, cultured, and imaged to decipher the prime sheet for stretch and culture conditions. PLCL sheets appeared to be premier and are awaiting imaging after being cultured with rat aortic explants. The goal is to see a gradual increase in SHG (for collagens) and/or 2PEF of elastin signatures over the coming weeks. Future studies with this system will focus on the mechanistic underpinnings of elastogenesis in various soft-tissue applications.

Acknowledgements (Optional):  This research was funded by NIH award R01HL157017 to Dr. Jonathan P. Vande Geest.

References
[1] Nivison-Smith, J., et al., Regenerative Medicine and Tissue Engineering: Cells and Biomaterials, 2011.
[2] Fernández-Colino, A., et al., Frontiers in Bioengineering and Biotechnology 7:340, 2019.
[3] Huang, A., et al., Tissue Engineering Part C: Methods 22:6, 2016.
[4] Almeida-González, F., et al., Materials Science and Engineering: C 120, 2021.
[5] Wanjare, M., et al., American Journal of Physiology-Cell Physiology 309:4, 2015.
[6] Di Francesco, D., et al. Biomolecules 13:9, 2023.