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  1. The Role Of Smooth Muscle Cell Contraction In Arterial Residual Stress Michael Mislan, Dr. Elena Di Martino Schulich School of Engineering, University of Calgary Funding provided by the Markin Undergraduate Student Research Program (USRP) in Health and Wellness Sense Mechanical Environment Extracellular Matrix Tissue Cells Abstract Objectives Results Change Mechanical Behaviour Residual stresses are present in mature arteries and serve the homeostatic function of equilibrating tensile stress over the arterial wall in response to physiological load. Arterial tissue is an open system comprised of constituents that are degraded and renewed at different rates. Residual stresses as well as mechanical properties are intimately related to the tissue microstructure. Any change at the microscopic level modifies the macroscopic behaviour and produces an adaptive response to restore the homeostatic condition. Prior research has found that the distribution of arterial stresses, both residual and global, depend on the mechanical properties of Collagen, Elastin, and Proteoglycans as well as Smooth muscle cell contraction[3]. However, many questions regarding how smooth muscle cells respond to the stiffness of their substrate in terms of morphology and phenotype have not been fully addressed experimentally. Therefore, the goals of this experiment were to investigate how smooth muscle cells affect and are affected by the mechanical behaviour of their extracellular matrix. • Isolate Smooth Muscle Cells from extracted tissue samples • Measure the stiffness of acellular and cell-seeded Collagen gels to determine how stiffness changes with Collagen concentration and smooth muscle phenotype • Determine how the smooth muscle cells’ cytoskeletal structure is affected by extracellular matrix stiffness • VSMCs were successfully isolated from extracted Rabbit aortas and cultured for multiple passages • Collagen mixture formulations were found that would produce a gel that can hold its’ shape • VSMCs were seeded into Collagen and remained viable long enough for macroscopic contraction of the gels to be observed Background ResidualStress Vascular Smooth Muscle Cells (VSMCs) Collagen • Collagen constitutes the bulk of Artery tissues’ mass • Consequently, Collagen is the main component of VSMCs extracellular matrix and its’ properties determines the VSMCs mechanical environment • Both Collagen fibres and VSMCs orient themselves in the direction of applied strain[4] • VSMCs attach to the surrounding Collagen fibres and contract, affecting the mechanical properties of the whole tissue • How VSMCs generate local strains on the Collagen substrate depends on their cytoskeletal structure • Residual Stress describes the intramural distribution of stress that forms in arteries during development • In the No Load State, the intimal and inner medial layers are in compression while the outer medial and adventitial layers are in tension • In the presence of external load (Blood Pressure), residual stress creates a homogenous distribution of stress over the thickness of the artery • The distribution of residual stress depends on the structural properties of Collagen, Elastin and Proteoglycans as well as Smooth Muscle Cell contraction • Vascular Smooth Muscle Cells (VSMCs) reside in the arteries' medial layer and are responsible for controlling dilation and constriction of the artery • VSMC phenotype can range anywhere along a spectrum between Contractile and Synthetic, and “spindled” and “spread” morphologies • VSMCs become progressively Synthetic in culture • VSMCs respond to external load and substrate stiffness phenotypically and morphologically • Phenotype modulation from Contractile to Synthetic is seen in many pathologies including Intimal Hyperplasia[2] and the formation of Aneuryms[1] Fig 6: Polymerized Collagen gel. • While the mechanical testing apparatus was completed, only the acellular collagen gels could be tested. Of the twelve gels that were prepared for this purpose, only a single gel was able to hold its shape after handling and mounting in the apparatus. • The imaging protocol was attempted, but technical problems prevented the capture of images. No Load State Loaded State Contractile Synthetic Fig 1: Intramural distribution of stress in the No Load and Loaded state, where Tensile is above and Compressive is below the radial axis. P is the internal Blood Pressure. Fig 7: Photograph of the testing apparatus with Collagen gel in blue. Fig 3: Biomechanical relationship between cells and tissue. Fig 2: VSMC phenotypic spectrum showing morphological differences. Methods Conclusions Smooth Muscle Cell Isolation Collagen Gel Experimental Design Progress was made in establishing the experimental methods including cell isolation, cell culture, cell seeding into the collagen gels and the stiffness testing device. More experiments are needed to reach conclusive results. • A Collagen gel was produced that could hold its shape and be handled • The final Collagen formulation included acid-solubilized Collagen, from rat tail tendons, 0.1M Sodium Hydroxide, 10x DPBS and DMEM • VSMCs were then seeded into these Collagen gels • Tissue samples of Rabbit thoracic aorta were extracted using aseptic technique • These tissue samples were enzymatically digested with a solution of Collagenase, Elastase, Bovine Serum Albumin and Soybean Trypsin Inhibitor to isolate VSMCs • The isolated VSMCs were then cultured and used between Passage 0-4 • Isolate VSMCs from extracted arteries and cultured • Design and build a novel apparatus to test the stiffness of Collagen gels • Measure how Collagen gel stiffness changes with concentration and investigate if stiffness changes with VSMC phenotype • Image the VSMCs cytoskeletal structure for cells seeded in Collagen gels of varying stiffness References [1] Ailawadi, G. Moehle, CW. Pei, H. Walton, SP. Yang, Z. Kron, IL. Lau, CL. Owens, GK. , 2009, “Smooth muscle phenotypic modulation is an early [2] Davies M.G., and Hagen P.O., 1994 “Pathobiology of intimal hyperplasia,” Br J Surg., 81, pp.1254–1269] [3] Rachev, A., and Hayashi, K., 1999, ‘‘Theoretical Study of the Effects of Vascular Smooth Muscle Contraction on Strain and Stress Distributions in Arteries,’’ Ann. Biomed. Eng., 27, pp. 459–468. event in aortic aneurysms,” J Thorac Cardiovasc Surg. 138(6), pp.1392-9. [5] Vader D, Kabla A, Weitz D, Mahadevan L. , 2009, “Strain-induced alignment in collagen gels,” Plos One 4(6):e5902 Mechanical Testing Fluorescent Imaging • The Elastic Modulus (E) of the Collagen gels was measured • The Collagen gels were mounted above a glass chamber that was pressurized with a manometer • A laser was used to measure the gels’ deformation • An analytical equation was then used to calculate E • F-Actin filaments of the VSMCs’ cytoskeleton were dyed with Oregon Green 488-conjugated Phalloidin • Fluorescent imaging was performed with an Olympus IX71 microscope equipped with Hammamatsu CCD camera and mercury tube fluorescent source Acknowledgements I would like to thank Dr. Walter Herzog and members of his research group for making available the Rabbits for their vascular tissue. I would like to think Dr. Donna Slater and members of her research group for help with the SMC isolation protocol and graciously providing us with advice and supplies. I would also like to thank Dr. Kristina Rinker and members of her research group for providing an excellent facility for cell culture, as well as invaluable advice and expertise. Fig 4: Schematic of the overall project broken down into the Cell Isolation and Microscopic and Macroscopic experiments. Fig 5: Apparatus schematic.