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A New Device for Mechanical Testing of Blood Vessels at Cryogenic Temperatures

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A New Device for Mechanical Testing of Blood Vessels at Cryogenic TemperaturesAbstractIntroductionExperimental ApparatusCryogenic Cooling SystemMechanical Testing SystemMaterials and MethodsTissue Model and CPATesting ProcedureCross-sectional Area MeasurementResults and DiscussionSummary and ConclusionsAppendixUncertainty AnalysisReferencesA New Device for Mechanical Testing of Blood Vesselsat Cryogenic TemperaturesJ.L. Jimenez Rios & Y. RabinReceived: 22 November 2006 /Accepted: 23 January 2007 / Published online: 27 February 2007#Society for Experimental Mechanics 2007Abstract As part of an ongoing program to study thethermo-mechanical effects associated with cryopreservationvia vitrification (vitreous in Latin means glassy), the currentstudy focuses on the development of a new device formechanical testing of blood vessels at cryogenic temper-atures. This device is demonstrated on a bovine carotidartery model, permeated with the cryoprotectant cocktailVS55 and a reference solution of 7.05M DMSO, belowglass transition. Results are also presented for crystallizedspecimens, in the absence of cryoprotectants. Resultsindicate that the elastic modulus of a specimen with nocryoprotectant, at about −140°C (8.6 and 15.5°C below theglass transition temperature of 7.05M DMSO and VS55,respectively), is 1038.8±25.2 MPa, which is 8 and 3%higher than that of a vitrified specimen permeated with7.05M DMSO and VS55, respec tively. The elastic modulusof a crystallized material at −50°C is lower by ∼20% lowerfrom that at −140°C.Keywords Blood vessels.Vitrification.Cryopreservation.Mechanical testing.VS55.DMSOIntroductionVitrification (vitro in Latin means glass) has been presentedas a promising alternative to conventional cryopreservation,whereby ice crystallization—known to be the cornerstoneof cryoinjury—is suppressed [1, 2]. Vitrification is achievedby means of rapid cooling of a highly viscous material (i.e.,cryoprotectant), when the cooling time scale is m uchshorter than the typical time scale for crystallization,causing the cryoprotectant molecules to be trapped in anarrested state. Vitrification can be achieved if the coolingrate exceeds a critical rate down to a specific temperaturethreshold, known as “the glass transition temperature.” Theglass transition temperature is cooling-rate dependent, andboth are intrinsic properties of cryoprotectant.The high cooling rate necessary for vitrification in largespecimens leads to a significant temperature distributionacross the specimen, at a point where the slowest coolingrate and the highest temperature are at its center. Hence, it isthe cooling rate at the center of the specimen that mustexceed the critical cooling rate in order to ensure thesuccess of cryopreservation. While the critical cooling rateis inversely p roportional to the cryoprotectant concentra-tion, cryoprotectants are potentially toxic, and the minimumconcentration required to promote vitrification is oftenapplied to a given thermal protocol. Seeking new cryopro-tectant cocktails that reduce toxicity effects, while increas-ing the likelihood of vitrification, represents an activeresearch area in the general field of cryobiology. Anotheractive area of cryobiology research is associated withpermeation techniques of the cryoprotectant into the spec-imen, either by diffusion, perfusion through the vascularsystem, or a combination of both.An undesired byproduct of rapid cooling is the develop-ment of thermo-mechanical stress, resulting from a non-uniform temperature distribution across the specimen.When it exceeds the strength of the material, thermo-mechanical stress results in structural damage [3] andfracture [4]. For example, immersion of frozen humanvalves directly into liquid nitrogen, for as little as 5 min,may result in tissue fractures [5]. This problem becomeExperimental Mechanics (2007) 47:337–346DOI 10.1007/s11340-007-9038-8J.L. Jimenez Rios:Y. Rabin (*)Biothermal Technology Laboratory,Department of Mechanical Engineering,Carnegie Mellon University,Pittsburgh, PA 15213, USAe-mail: [email protected] when a hospital-based frozen valve storage systemoverfilled during an automatic refill cycle. Valves from thisaccident were discovered to have numer ous full thicknessfractures in the valve conduit, following normal thawingprocedures in the operating room [6]. Adams et al. [5]reproduced this phenomenon experimentally.The thermal expansion of frozen biological materials––which is the driving mechanism of thermal stress––hasbeen intensively studied in recent years, both in crystallized[7] biomaterials (relevant to classical cryopreservation) andvitrification [8–12]. The current study is aimed at exploringthe mechanical response of frozen biomaterial in typicalcryopreservation protocols. The cryoprotectants applied inthe current study are dimethylsulfoxide (DMSO) and thecocktail VS55, where their specifications are described inthe “Material and Methods” Section below , and a reviewof their development and application for cryopreservation isgiven in [1]. The current report describes a new device formechanical testing in typical cryopreservation conditions.Finally, the current report pre sents typical results f orcrystallized blood vessel specimens, and vitrified bloodvessel specimens below their glass transition temperature,where the response of the material can be characterized asthat of a solid over the testing time period.Experimental ApparatusTwo objectives have been set forth for the design of theexperimental apparatus: (1) to enable repli cation of atypical cryopreserva tion protocol on the specimen prior tomechanical testing, while it is attached to the mechanicaltesting device; and, (2) to enable holding the specimen at apre-specified cryogenic temperature for an extended periodof time thereafter, over the course of mechanical testing.Those objectives correspond to the two phases of experi-mentation, respectively: mimicking a cryopreservatio nprotocol while (despite the thermal contraction), the spec-imen is maintained free of external load (Phase I), andstress–strain measurements at a constant cryogenic temper-ature (Phase II). With reference to Fig. 1, the experimentalapparatus is compri sed of two systems: a mechanicaltesting device, and a cryogenic cooling system, which aresequentially described below.Cryogenic Cooling SystemThe cryogenic cooling system is a modification of aprevious experimental apparatus, designed for


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