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Mechanical properties of biomaterials are difficult to characterize experimentally because many relevant biomaterials such as hydrogels are very pliable and viscoelastic. Furthermore, test specimens such as blood clots retrieved from patients tend to be small in size, requiring fine positioning and sensitive force measurement. Mechanobiological studies require fast data recording, preferably under simultaneous microscope imaging, in order to monitor events such as structural remodeling or localized rupture while strain is being applied. A low-profile tensile tester that applies prescribed displacement up to several millimeters and measures forces with resolution on the order of micronewtons has been designed and tested, using alginate as a representative soft biomaterial. 1.5% alginate (cross-linked with 0.1 M and 0.2 M calcium chloride) has been chosen as a reference material because of its extensive use in tissue engineering and other biomedical applications. Prescribed displacement control with rates between 20 μm/s and 60 μm/s were applied using a commercial low-noise nanopositioner. Force data were recorded using data acquisition and signal conditioning hardware with sampling rates as high as 1 kHz. Elongation up to approximately 10 mm and force in the range of 250 mN were measured. The data were used to extract elastic and viscoelastic parameters for alginate specimens. Another biomaterial, 2% agarose, was also tested to show versatility of the apparatus for slightly stiffer materials. The apparatus is modular such that different load cells ranging in capacity from hundreds of millinewtons to tens of newtons can be used. The apparatus furthermore is compatible with real-time microscope imaging, particle tracing, and programmable positioning sequences.

Efficient haemorrhagic control is attained through the formation of strong and stable blood clots at the site of injury. Although it is known that platelet-driven contraction can dramatically influence clot stiffness, the underlying mechanisms by which platelets assist fibrin networks in resisting external loads are not understood. In this study, we delineate the contribution of platelet-fibrin interactions to clot tensile mechanics using a combination of new mechanical measurements, image analysis, and structural mechanics simulation. Based on uniaxial tensile test data using custom-made microtensometer, and fluorescence microscopy of platelet aggregation and platelet-fibrin interactions, we show that integrin-mediated platelet aggregation and actomyosin-driven platelet contraction synergistically increase the elastic modulus of the clots. We demonstrate that the mechanical and geometric response of an active contraction model of platelet aggregates compacting vicinal fibrin is consistent with the experimental data. The model suggests that platelet contraction induces prestress in fibrin fibres, and increases the effective stiffness in both crosslinked and non-crosslinked clots. Our results provide evidence for fibrin compaction at discrete nodes as a major determinant of mechanical response to applied loads.

Understanding clot biomechanics is critical for the treatment of cardiovascular diseases. Based on our recent observation that that the structural configuration of the clot network correlates well with the mechanical properties such as stiffness, we hypothesized that the heterogeneity in the mechanical response of the microstructure dictates clot micromechanics and hence the macroscopic behavior. To test this hypothesis, we have custom-developed a microextensometer device coupled to a microscope to probe and image microstructural changes and micromechanical behavior of fibrin and blood clots. 20 μL clots were pulled at a prescribed strain rate of 60 μm/s using a programmable nano-positioner, and the force was measured using a 10 g load cell and acquired at 500 Hz. From the stress-strain measurements, we observed that both FFP and blood clots showed non-linear and abrupt changes in resistive tensile force in response to constant strain rate (Fig. 1A). Using fiduciary markers, we observed that cross-linked, but not uncrosslinked, fibrin clots showed a microscopically non-uniform deformation in response to macroscopically constant strain rate. Further, computational analysis of the mechanical response of clot microstructure to an applied stress revealed heterogeneity in strain energy distribution dictated by the network properties (Fig. 1B). Together, our results suggest that the heterogeneity in microscale translates to the non-linear response at the macroscale, and will ultimately dictate the pathophysiology of thrombosis.

NONLINEAR STIFFNESS AND VISCOELASTICITY OF INHIBITOR-TREATED BLOOD CLOTS BY TENSILE TESTING by Wilson S. Eng Although blood clots are vital to wound healing, little is known about what factors influence clot stiffness and dynamic response. This work investigates the mechanics of inhibitor-treated clots by direct tensile testing using a custom designed system for forces below 1 N. Inhibitors that affect clot formation include blebbistatin, which affects myosin II movement on actin, and cytochalasin D, which affects actin polymerization. The hypothesis of this investigation is that blebbistatin will have a greater effect on mechanical behavior than cytochalasin D, because the inhibition of myosin II will weaken the overall clot more than actin. This hypothesis was investigated using clots that were treated with blebbistatin and cytochalasin, using untreated whole blood as a reference. Clots were tested from five different donors with at least two replicates from each donor. Each clot was subjected to an initial stretch ratio of 1.5 to measure nonlinear stiffness, followed by a series of 1 mm increments to record stress relaxation. At a stretch ratio of 1.5, blebbistatin-treated clots exhibited 4.3% lower tensile stress than cytochalasin-treated clots. The relaxation time constant for blebbistatin-treated clots was 10% faster than for cytochalasin-treated clots. This evidence supports the hypothesis about the role of myosin II in blood and introduces experimental methodology that can be extended to studies on mechanics of other soft biological tissues. v ACKNOWLEDGMENTS This work was supported in part by the Kordestani Endowment to the Charles W. Davidson College of Engineering at San José State University (SJSU) and in part by the AHA Institutional Research Enhancement Award (AIREA) program of the American Heart Association (Award Number 18AIREA33960524). I would like to thank Dr. Anand Ramasubramanian for providing background on the project and direction needed in testing blood clots. I would like to thank Dr. Amit Saha for his support with the specimen testing development. I would like to thank Dr. Sang-Joon (John) Lee for tirelessly providing guidance throughout the entire project and reviewing. I would like to thank members of the MEMS Lab at SJSU for help with fabrication and testing. Finally, I would like to thank my friends and family for their support. vi TABLE OF CONTENTS List of Tables .