,

"
.

Proceedings the of

ASMFJJSMEJoint Thennal EngineeringConference March 15-19,1999,SanDiego, California

REA T TRANSFERIN BIOMEDICAL ENGINEERING AND BIOTECHNOLOGY

Boris RUBINSKY Department MechanicalEngineering of University of Califomia at Berkeley BerkeleyCA 94720. USA rubinskv@newon.berkelev.edu

Keywords: bioheat transfer, thermal biological sytstems.thermal evolution, hyperthermia, hypothermia, cryosurgery.cryobiology

ABSTRA CT
Heat transferplays an important role in biological systems.The goal of this article is to introduce researchersin the general area of engineering heat transfer to the area of bioheat transfer.To this end I will review selected topics from eachof the different areasthat comprisethe field of biological heat transfer. Prior to a discussionof the heat transfer in organismsthat exist on earth now, I will introduce the area of thermal evolution. Than I will describeselected topics of research in each of the following temperatureranges:a) temperatures higher than the physiological temperatureof warm-blooded organisms,from about40 c to about 120c, b) temperatures the physiologicalrangeof warmin bloodedorganisms,from about36 c to about40 c and c) temperatures lower than the physiological temperature of warm-blooded organisms,from about 36 c to - 273 c. In the

first rangeof temperaturethe review focuseson the use of high temperatures medicine to treat in undesirabletissue and on the study of thermal accidents.In the second range of temperatures the review focuses three scales:a) microscale, on in which I win discuss molecular motors, b) mesoscale in which I will discuss the heat transfer between blood and tissue and c) the macroscalein which I will discuss the area of whole body heattransfer. The third temperature range can be subdivided into two; temperatures above freezing and below freezing. Temperatures this rangeare important because in they can be usedto preservebiological tissueand cells and because they can be also used to destroy undesirabletissue. Here I will discuss the thennal mechanismsof cell damage in this temperature range and also cold and freeze tolerantanimals.

.~ ..'

) \

INTRODUCTION Life has evolved in relation to the temperatureand the thermal environmentof our planet. It is no coincidencethat the optical nerve is affected by radiation only in the narrow wavelength range of between 0.38J1m and 0.76Jun. This range, known as visible light, correspondsto the peak of solar radiation. It is also no coincidence that all warm blooded animals have a deep body temperature of between36 c and 40 c. This temperature 15 is c above the average temperate zone temperatureof 22 c. The cooler environment servesas a heat sink to warm-blooded organisms and allows them to function effectively at a constant temperature, while continuously producing heat. From thermal considerations, it is obvious that warm blooded animals, which have an aerobic metabolism,continuously bum oxygen and produce heat. could not maintain their current temperatureif the temperatureof the environment would have been 40 c, for instance.In a different thermal environmentwe would have evolved differently, and as the thermal environment of our planet changes terrestrial organisms change. In studying the broad area of bioheat transfer I find that the research on life processesin relation to the current thermal environmentof our planetcan be divided into threetemperature ranges: Temperatureshigher than the physiological temperature of warm-blooded organisms, from about40 c to about 120c. Temperaturesin the physiological range of warm-bloodedorganisms,from about 36 c to about40 c. Temperatureslower than the physiological temperature of warm-blooded organisms, from about36 c to - 273 c. Distinctly different bioheattransferproblemsare encounteredin each of these three temperature ranges. The goal of this article is to introduce researchersin the general area of engineering heat transfer to the area of bioheat transfer. Therefore in this paper I will describethe topics only briefly and the goal will be to introduceas many topics as this spacepermits. I will discuss researchtopics and problems typical to each of the three temperature rangesof relevance to bioheat transfer listed above. However, before I proceedwith a discussionon the bioheattransfer problems in organisms that live in the current

thermal environment I want to address briefly the exciting topic of thermal evolution

THERMAL EVOLUI10N Origin of lite One of the most exciting questions in the history of scienceis the origin of life. Has life originatedin a cold or in a hot environment? Haslife originatedin the watecsof hot springs or geothermal ventsor in cool poolSor cold oceans. Proponents the hot origin of life point to the of fact that many of the organismsthat Populatethe earliestbranchesof the tree of life live today in extreme thermal environmental conditions (Forterre, 1996; Vargas et aL, 1998; Vogel, 1999). These organisms, known as hyperthermophiles, thrive between80 c and 90 c. Supportfor the proponents the cold origin of of life hascomes from a recent study by Galtier et at. (1999). GaItier and his colleagues have approached questionabout the thermal origin the of life from a molecularperspective.They have tracked the evolution of two temperature sensitiveribosomeRNA (rRNA) moleculesfrom bacteriato mammals. (The RNA is composedof nucleotidebasesthat form temperaturesensitive bonds. Some of the bonds withstand high temperatures better than others. The bases guanine(G) and cytosine(C) form a strong bond while adenine(A) and uracil (U) form a weaker bond. Studieshave shown that the rRNA of heat loving organismshasmore G and C than A and U, presumablebecausethe G-c bond is more stableat higher temperatures than the weaker AU bond (Vogel, 1999).)Using a Markov model, GaItier et at. (1999) have evaluated the G-c nucleotide content of rRNA in the organisms whoseevolution they have tracked.Their results show that "The inferred G+C content of the common ancestor to extant life forms appears incompatiblewith survival at high temperature". The ancestral organisms apparently lived at moderate temperatures. With respect to hyperthermophilicspeciesthey favor the notion that "extant hyperthermophilic species evolved from mesophilic organisms via adaptation to high temperatures" Altho~gh much exciting . work is being done in studying the thenna1 origin of life, the answersare not yet conclusive. Both the molecular and the cellular researchinto the origin of life are open areas of considerable interest to the heat transfer community. Scientists from the heat transfer and thermodynamic analysis community could contribute from their perspectiveand training to the searchfor the thermalorigin of life.

..-

Origin of endothermy Another interestingquestionin thennal evolution relates to the origin of endothermic organisms.Severalbooksand reviewshave been written on this topic (Ruben, 1996; Fastovsky, 1996; Randolph, 1994). Depending on the method of thennoregulation, organismcan be an classified as either endothermicor ectothennic. Endothermic organismsgenerateheat from the body while ectothennic ones obtain heat from their environment Maintaining a constantbody temperature is essential to the survival of endothermic animals. Enzymes in endothermic organisms, for example, denature during hyperthermia, when the body temperaturerises above 40 C. The first organ to be affected by temperaturesabove 40 c is the brain. Serious disruptions leading to deathensueif the brain is exposed to high temperatures, a prolonged for period. On the other hand, hypothermia(lower than normal body temperature) causeequally can disastrousresults in slowing metabolicactivities and impairing brain function. In contrast to ectothenns,which control their body temperature mostly by behavioral adaptation, endotherms maintain it by physiologicalmeans, throughtheir aerobic metabolism. Endothermic animals require more energy expenditure than ectothennic ones since they have to maintain their constant temperature elevated over the average environmental temperature. Large amounts of energy are spent by endothennson thermoregulation and this seems to suggest inefficiency of maintaining such a high metabolic rate. Moreover as more time is spent on obtaining food, there is also higher risk of exposure to danger such as predators and weather hazards. What are the origins of endothenny? Speculationshave been made on the origin of endothenny as the selection of a high stable temperature for important life processes such as enzymatic catalysis, independenceof daily timing and resistanceto freezing. However,thermoregulation alone may not justify its evolution. Metabolic increment cannot have solely come from attempt to attain thermoregulationalone since in the initial stages of the evolution of endothenny,the mechanism would not have been effective in attaining thennoregulation. In addition endothermymay have first evolved during the Mesozoicwhich is one of the most thermally stableperiodsin earth history. Thermodynamic studies were made which compared oxygen consumption of ectothenns and endotherms during different activities. These studies suggest that the

increased aerobic metabolism of endotherms over the anaerobicmetabolismof ectotherms is the main factor in the evolution of the endotherms. Even thoughthe cost of maintaining a high aerobicmetabolicrate is significant. there are also a numberof advantages that offset the cost. Sustainedstaminacan assist herbivores to escape carnivores and help predatorsin capturing their pray. The key step in evolution may have been the elevation of metabOlic rates with aerobic respiration and thermoregulation may have been a consequence the elevation of of metabolic rate with increased aerobic metabolismcapacity. The study of the evolution of endothermyis anotherexciting topic of heat and mass transfer that should be studied by researchers heattransferand thermodynamics. in TEMPERATURES IN THE PHYSIOLOGICAL RANGE OF WARM BLOODED ORGANISMS Understandingthe thermal behavior of warm-bloodedorganisms(endothenns)can lead to a better understandingof life processesin general and has applications in health care, engineering for human comfort and medicine. The study of heat transfer in warm-blooded organismstakes place at three different length scales:molecular(microscale),whole organsand tissues (mesoscale) and whole body (macrosca1e). the molecular scales there is At extensive research in the areas of thermodynamics proteinsassembly,molecular of conversion(photosynthesis, ATP (adenine three phosphate)production and molecular motors), ionic pumps (active transport of ions) and cellular metabolism (efficient combustion at room temperature). At the mesoscalethere is interest in understandingheat transfer between blood vessels and the surrounding tissue, measuring blood flow with thermal clearance, understandingthe thermal effect of malignant tissue, respiratory heat transfer, and even heat transfer in teeth. The thermal macroscale researchbas been the realm of the traditional bio-thermal engineer. Extensive work was done in designing thermal comfort systems (beating, air conditioning and thermal comfort), studying thermoregulation, heat transfer in extreme environments(either in space,deep in the ocean, or during fires), developingan understandingfor the heattransferin newborn,elderly and sick. In the following sections I will introduce a few examplesof the thermal work in each of these three scales.

Thermal processes at the microscale Biological systems produce energy. store energy and convert energy to work. This is what any traditional inanimate thermal energy conversion system does. through various processes of combustion. energy storage and mechanical motors and pumps for converting the energy to work. However. there is a major difference between biological systems and inanimate systems. Biological energy conversions systems are true nanodevices. which function at constant temperatures of about 37 c to 40 oCt with superior efficiency. In contrast inanimate energy conversion systems are macroscale device in which the combustion process takes place hundreds of degrees above the body temperature with lower efficiencies. Nature has designed compact motors known as proteins (kinesin's motor domain is 350 amino acids) that convert chemical energy at room temperature from adenine triphosphate (ATP) hydrolysis to work with an efficiency as high as 50% in the case of the myosin molecule. (Vale. 1993). Protein motors produce muscle contraction, cause organelle motion and cell motion. Attempts are currently made to understand how these motors work and to design thermodynamic models for these motors. These proteins work like stepper motors and a typical design can be found in Vale (1993). Recently. Kitamura et al. (1999) have used a scanning probe with a fluorescent label to detect that myosin moves along an actin filament with single mechanical steps of approximately 5.3 nanometers. Protein motors have also other functions. They activate ionic pumps across cell membranes. Woiling in reverse these protein pumps can use trans-membrane ion gradients to manufacture ATP, the fuel of the biological system. Probably some of the most intriguing studies in this field are by the group of Oster. (Wang et al., 1998; Elston et al.. 1998; Elston et al.. 1997; Doering et al.. 1995. For instance in Elson et al. (1998), they have developed an equivalent thermodynamic and mechanical description of a proton pump and engine. They describe how a flow of protons. driven by the difference in Gibbs free energy across the mitochondrial membrane can produce three ATP's per twelve protons passing through the motor. 1be motor protein known as ATPase is shown to be composed of two counter-rotating assemblies, one a stator and the second a rotor. 1be motor must generate sufficient torque to produce three A TP's per revolution, an amount of work equi valent to 20 lea per ATP molecule. T

(ka is the Boltzman constant and T is the absolute teMperature;at room temperature the value is 0.6 kcal mol -1 ). Elson et at. (1998) show that the motor is reversible and can act both as a pump and as a meansto convert ionic flow into stored useful energy, the A TP molecule.While the goal of modem engineering is to design more efficient energy conversion systems and to build smaller and smaller micromechanical systems,it is difficult to ignore the fact that suchsystemsalreadyexist in nature. Thermal processes the mesoscale at Probably the most extensive body of work produced by the bioengineering heat transfer community is in the mesoscale. Much researchhas been dedicated to modeling heat transferin biological systems. Tissue is different from other materialsbecause the presence a of of vascular system and its associatedblood flow, metabolic heat transfer and heterogeneous thermal properties.The effect of blood flow is mostdifficult to accountfor and researchers have proposed different models. More detailed reviewsof bioheat transfer models can be found elsewhere(Chato, 1981; Shitzer and Eberhard, 1985;Baish et aI., 1986; Chamy, 1992; Eto and Rubinsky, 1996). The most widely usedthermal model is commonly known as the bioheat equation (pennes, 1948). This formulation includes a source/sinkterm, which accountsfor the heat transfer from blood perfusion. The general formulation is,
aT. . -T,,) P,C'T=V(k,VT)+q_,+liJpbCb(T.

at,

where p is density, c, heat capacity, T, temperature, time, k, thermal conductivity, m, t, volumetricblood flow and the subscripts.t, stand for tissue,b for blood, a for arterial blood and v, for venousblood. The bioheat equation assumes that all the thennal effects of the blood can be absorbed in the last term of the equation, a sourceterm. Here it is assumedthat blood enters any volume at tissue at the arterial temperature and leaves at the venous temperature while transferringall the blood energy instantaneously. Although it is widely used, the equation has shortcomings. One difficulty is the determination of the blood perfusionrate, and the fact that the blood flow is given a scalar term. when in fact it is a vectorial quantity. There are other models. The Wulff (1974) model describesthe tissueas a porous media and attributes the blood a Darcy' velocity. Chenand Holmes (1980) expandon the

Pennes equation and add two new terms including the Darcy velocity in a porousmaterial and a term that describes the directional enhancedheat transfer due to the blood flow. Weinbaumand Jiji, (1985) developeda seriesof new bioheat equationsthat originally dispensed with the volumetric blood flow term and included only the directional flow of blood and recently developed a new significance to the volumetric blood flow term. The need to analyze biological heat transfer at the blood vessellevel hasgenerated a series of mathematical models describing the direct heat transfer interaction between blood and tissue.Chen and Holmes(1980) wereamong the rust to perform a simple energybalancefor a single blood vessel embedded in tissue. The solution is a simple exponentialthermal decay, typical of heat exchangers. analyzing various In typical blood vesselparameters they recognized that only blood vesselslarger than about 80 J1111 are thermally significant, while smaller blood vesselsare not. Chato (1980) performedat the same time a similar analysis treating the tissue and the blood as heat exchangers while Weinbaumet at. (1984) approached problem the as an entry length problem. In tissue, blood vesselswith diameterslarger than the thermally significant blood vessels occur as vein-artery pairs. Mitchell and Myers (1968) were the first to analyze the local heat counter-currentheat transfer, between the arterial and venous blood flow in the pair. This counter current heat transfer is one of the most important aspectof heat transfer in biological systems at the mesoscale.
The area of bioheat transport has witnessed in the last two decades,increased activity. Much of the work is related to developingmathematical modelsfor the effect of blood flow on heat transfer in tissue.This work was triggered by the interest in hyperthermia treatment of malignant tissues, a topic I will discusslater. While many advanceswere made in modeling heat transfer in tissue, this area is not sufficiently understood.

transfer and what is considered to be thennal comfort. A ~ood summary of the field can be found in the ASHRAE Fundamentals (1997). The worlc in this field includes: evaluation of what constituteshumanthermal comfort, studies on the effect of clothing on heat transfer to the human body (Xu and Werner, 1997), mathematicalmodeling of heat transfer in the whole humanbody (Yokoyamaet al, 1997).The mathematical models of the' human body describe segmentsof the body as a lumped thermal mass and were developed first by Stolwijk and Hardy (1966). Currently the research focuseson expandingthesemodels into smaller thermal compartmentsand developing models for thermoregulation. Improved experimental thermal mannequins are being designedto measurethe heat transfer between the body and the environment (Holmer and Nilsson 1995). Studies are also carried out in such areas as office and vehicle microenvironment thermal control and thermoregulation. There are important applications of this field in medicine. Heat transferand temperatures important in sports are medicine(Hargreavesand Febbraio, 1998) and in anesthesiology(Saito et aI. 1998). Another importantapplicationrelatesto the changein the thermal behavior of humans with aging. The thermalbehaviorof humanschangeswith age in both the newborn (Benedict and Talbot, 1915) and the elderly (Shock. 1961; Florez-Duquet,et aI. 1998).It is important for the bioheat transfer community to worlc in the area of medical research. Temperatures and heat transfer playa significant role in disease and dead, particular in the extreme age groups, (O'Neal et ai, 1984; Kenney and Havenith, 1993), and many of the engineeringaspectsof the medical heat transfer areahavenot beenresolved. TEMPERA WRES LOWER THAN THE PHYSIOLOGICAL TEMPERATURE OF WARM BLOODED ANIMALS. Low temperature heat transfer is affectedby the fact that water is the largest tissue component Water freezes at 0 c and the behavior of the biological system changes entirely when the water is removed from the systemas inert ice. Therefore low temperatures biological heat transfer can be divided in two temperature ranges: temperatures above the freezing temperaturefor water and temperatures below the freezing temperature water. Here I for will discussthe behavior of biological systemsin

Thermal processes the macroscale at Currently most of the industrial applications of bioheat transfer are in the macroscalerange. These relate primarily to the design of heating, ventilation and airconditioning systems. The design requires a good understandingof the whole body heat

these two temperatureranges in two separate sections. Biological systems in the temperature range between normal mammalian physiological and 0 GC. Studying life in the temperaturerange of betweenthe normal physiologicaltemperature of warm blooded animals and the change of phase temperature of water has practical applications in developing methods for preserving mammalian cells and tissues by refrigeration. Cell and tissue refrigeration is important in biomedicineand biotechnology.I will begin with a discussionof phenomena that occur when the temperature of biological systems are reduced from their normal temperature and thanI will discussorganismthat haveevolved the ability to live under conditions of low temperatures.Understandinghow these organismssurvive may lead to the development of techniques preservation cells and organs for of by refrigeration Changesin biological systemsduring cooling. Cells are entities in which an intracellular solution is separated from the extracellular milieu by a membrane. The intracellular composition is entirely different from the extracellular composition. Cell and subcellular membranesmake life possible by isolating the interior of the cell and subcellular components from their exterior. Thereby, membranes preserveand control the cell identity in the face of uncontrollable changes in the environment. The cell membrane acts as a barrier. Since the interaction and the mass transfer betweenthe interior and the exterior of the cell is important, the cell membrane also acts as a gate that regulatesthe masstransfer.This is done through a variety of channels,pumps and other methodsof controlled mass transfer. The cell membranealso servesas an anchor to the cytoskeletonthat providesthe structuralintegrity of the cell. Cell membranes are made of phospholipidsarrangedin a bilayer configuration in which the hydrophobicheadsof the molecules face outward of the bilayer and the hydrophilic tails face inward. This configuration producesa membrane that is impermeable water.The cell to membraneis than spanby transportproteinsthat form channelsand pumps.There is a very large variety of different molecular species of membrane phospholipids.

In a nonnally functioning cell, lipids (fats) are in' a liquid phase. However, lipids undergophasetransition, upon cooling. This is the familiar phenomenon fat solidifying in a of cooking pan after it cools. The cooled lipid bilayer can undergo several different types of phase transfonnation. Some of the new structures that fonD are known as inverted micelles,hexagonalII structuresor cubic phases Siegel,(1994). A detailed review of the process of lipid phasetransition can be found in Quinn (1985). Studyingthe phasetransition in the lipid bilayer is an interesting area of research within the realm of the heat transferengineer. Some of the most common methods for studying these phase transformations utilize the optical properties of membranes, particular in the infraredrange(Tablin, F et aI., 1996). During cooling, important changes occur in the cell membranestructure and in the cell membranefunction (Drobnis, E Z et al. 1993). The membrane lipids undergo phase transition as the cell cools. 1be new lipid configuration fails to anchor tightly the membraneproteins and new openings form in the cell membrane.The membrane looses the ability to isolate the cell interior from the exterior. Uncontrolled mass transfer begins to take place betweenthe cell interior and exterior, which leadsto cell damage(McGrath, 11. 1997). The membrane proteins and the binding between the cytoskeleton and the membrane are also affectedby cooling. 1be masstransferactivity of the membrane proteins involves chemical reactions related to the A TP molecule. A reduction in temperature reduces the thermodynamicefficiency of these membrane proteinsand further reducesthe ability of the cell membrane to regulate the intracellular composition. The bonds between the cell membrane and the cytoskeleton are chemical bonds that are temperature dependent. As the temperature reducedthese bonds weaken and is the cytoskeleton fails. Obviously all these temperature relatedphenomena causedamageto a mammaliancell that is cooled to temperatures lower than the normal physiological temperature. How can a refrigerated mammalian cell be protectedfrom thesemodesof damageand how can one preserve cells in a refrigerated state? Nature has found solutions. Some of them we just begin to understandand some are not yet understood. Probably the animal model most relevant to the study of refrigeration of mammaliantissueis the hibernator.

Hibernators Mammals normally maintaintheir body temperature constant during the cold winter months by increasing their metabolism to compensatefor the increasein heat loss and by adaptationchanges. Adaptationchangesinclude additional layers of insulation on the body surface or habitat changes. When a warm bloodedanimal temperature reducedbelow the is physiological temperature it will eventually loose its ability to control the metabolismand die. However some mammals,the hibernators, have evolved mechanismsthat allow them to survive at low temperature. There are a number of differences between obliged homeotherms (regular mammals) and hibernators.When an obliged homeothermis artificially cooled below 20C to 25 c all measurable signsof brain and nervous activity cease. The hibernators, however, lower their core temperature a can to few degreesabove the ambientand enter a state of torpor. The hibernators' brain continues to function at these reduced temperatures.Most mammals retain a relative constantheart rate as their temperatureis lowered, until they reach a temperature irreversibledamage. contrastin of In hibernators there is a gradual decrease heart in rate with temperature. The heart of the hibernators can maintain rhythmic beating at temperaturesnear 0 C. As the environmental temperature drops, an obliged homeothermic mammal will attempt to retain a constant high physiological core temperature.Unlike obliged homeotherms, the hibernator thermoregulatory mechanism allows the animal to retain a thermoregulatedtemperature, at a level that but is significantly reducedand close to that of the environment. Therefore the heat loses in a hibernator are much smaller than in a mammal under the same thermal conditions.Because the heat lossesare small the hibernatorcan survive the long winter months without metabolic heat generationand without the consequent need for food. Obviously hibernatorscan survive at low temperatures. But how can the hibernators survive the fatal changethat cells experienceat low temperatures? The mechanism related, at is least in part, to molecular changes.Obviously maintaining a fluid membrane at lower temperaturesis essential. It appears that in hibernators the cell membrane lipid phase transition temperature is reduced during hibernation.This is accomplished changesin by the lipid compositionof the cell membrane either prior to entering hibernation or throughout the

period.The secondmechanism may relate to the protein mas~ transfer carriers across the cell membrane. Apparently in hibernators at low temperatures, while the metabolism is reduced the activity of the ionic pumpsand the ability of the mitochondriato produceA TP is maintained. The fundamental mechanisms by which hibernators achieve survival at reduced temperature not yet fully understood. 'There are is an extremely small community of scientists working in this exciting bio-thermal areaand the engineering heat transfer community can contributeto this research. good review of this A area can be found in several books (Wang, 1982);Carey,C., et at. (Ed.), 1993). Antifreeze proteins and trehalose In response to low environmental temperatures,many fish and plants produce proteins with special properties.These proteins are known as antifreeze proteins. There are several reviews on these proteins and their properties (Ananthanarayanan,1989). Here I will addressonly their low temperaturerelated properties.In a later section I will discuss their freezing related properties.Studies have shown that antifreeze proteins protect the integrity of the oolema (membrane)in oocytes (unfertilized eggs) at low temperatures(Rubinsky, et aI. 1990), they reduce ionic leakag~ from the cell membraneat low temperature(Rubinsky et aI., 1992) and inhibit ion transport across the cell membrane (Negulescu et aI., 1992). It is conceivablethat theseproteins have evolved to inhibit the mass transfer acrosslow temperature impaired cell membranes (Rubinsky et aI., 1991).Studieswith liposomesand platelets have shown that while the antifreeze proteins inhibit masstransferacrossthe cell membrane they do not modify the lipid phasetransition of the cell membrane(Hays et aI., 1997; Hays, et al. 1996). Because antifreeze proteins block ion transport they were successfully used in organ preservation during hypothermia and in preservation such difficult cells to preserve,as of platelets.In addition to antifreezeproteins it has been also shown that the sugar trehalose has beneficial propertiesfor preservation cells in of a hypothermic state (Ring and Danks 1998). Trehalose is being produced by organisms in nature(Singer, et aI. 1998),and itwas shown to stabilize the cell membraneagainstcold damage (Beattieet aI., 1997).

.'
BIOWGICAL SYTEMS AT TEMPERATURES LOWER THAN THE FREEZING TEMPERATURE. Freezing of biological systems is another areaof biological heattransferto which scientistsfrom the heattransfercommunity have made major contribution. Freezing has a paradoxicaleffect on biological systems.On one hand freezing can be usedto preservebiological materials and on the other hand freezing can destroy biological material. The preservation of biological materialsin a frozenstateis known as cryopreservationand the controlled destruction of biological materials is known as cryosurgery and is used for treatment of cancer and other undesirable tissues. To successfully either preserve or destroy biological materials by freezing in is important to developa fundamental understandingof the processes occur during that freezing. There are severalextensivereviewsand books on the fundamentals and the use of freezing in cryosurgery and cryopreservation (pegg and Karrow, 1987; McGrath and Diller, 1988; Diller, 1992; Onik et at. 1994; Diller 1997; Rubinsky, 1998). Here I will review briefly some of the fundamentalaspectsof the field. Studying the process freezingof cells of in a cellular suspension and in tissue has identified several universal mechanisms of damage.It was observedthat when a biological medium begins to freeze, freezing usually starts in the solution that surrounds the cell. The volume of the extracellular solution is much greater than that of any cell and therefore the probability of extracellularsolution nucleationis much greaterthanthat of intracellularnucleation. When the extracellularsolution beginsto freeze the two phasemixture of ice and solution is in a state of thermodynamicequilibrium. In contrast, the unfrozen solution inside the cells is thermodynamicallysupercooled. Therefore,there is a difference in Gibbs free energybetweenthe solution inside the cell and that outside.The cell membrane has a greater permeability to water than to other solutes.Therefore,to equilibratethe difference between the intracellular and extracellular Gibbs free energy water will leave the cell. As the cell dehydrates, the concentrationof solutesinside the cell increases. The increasedintracellular concentrationcauses chemical damageto intracellularproteins.This is the first mode of damageduring freezingof cells in solution or in tissue.The chemicaldamage a is function of concentrationand temperature. When the dehydratingcells are rapidly cooled to lower temperatureS chemical damage is reduced. the The higher the cooling rate (rate of temperature change) during freezingthe shorter the period of time cells are exposedto the lethal combination of high intracellular concentration and high subzerotemperatures shorter periods of time. for Therefore, an increase in cooling rate will increase cell survival. However, water transport across the cell membrane is time dependent Consequently, when the cooling rates are high enough the intracellular solution will be sufficiently supercooledto nucleate and freeze rapidly intracellularly. This is the second mode of damageduring freezing. The two modes of damage described above occur in cells in solution and in tissue. A typical cell survival curve that depicts cell survival as a function of cooling rate hasan inverseU shape.Cell survival is low first at low cooling rates,increases toward an optimum with an increasein cooling rates and thandropsas highercooling rates that lead to the formation of intracellular ice are reached.Each cell has its own survival curve. These curves dependon the cell size, membranepermeability and intracellular nucleation sites. Thece are additional modesof freezing damage. Recently Ishiguro and Rubinsky (1994), (1998) have shown that cells are also damaged by the mechanicalinteraction betweenextracellular ice and the cell. In addition, in tissue, the dehydrationof the cells results in a defonnation of the extracelular matrix, destruction of vasculatureand damage to the functionality of the organ. Equationsdescribing the process of freezingin cells and tissuescan be found among other referencesalso in (Rubinsky and Pegg, 1988; Rubinsky, 1989; Bischoff and Rubinsky, 1993). Experimentally the most important tool for studying freezing is the cryomicroscope. A review of the history of cryomicroscopy can be found in McGrath and Diller (1988). Mechanisms of freeze avoidance and freeze tolerance in nature. In nature, many cold-blooded animals and plants are exposed to temperaturesbelow freezing. How do theSe'organisms survive. Apparently, there are two mechanisms, freeze avoidanceand freezetolerance. Freezingcan be avoided by reducing the change of phase tempecature the biological solution. One of of the mechanismsto reduce the change of phase temperatureis colligative i.e. by increasing the concentrationof solutesinside the tissue. There are animals that during winter generate high

"

concentrations of intracellular solutes, such as glycerol (Storey, 1997). Certain insects can produce in winter high, molar concentrations of glycerol (Storey, 1997; Duman, 1991). While avoiding freezing, the high concentrationsof glycerol also stops the life processesin these organisms, making them extremely susceptible to destruction. This mechanism of freeze avoidanceis effective in insectsbecause their of large numbers. Other organisms,such as deep ocean fish, survive in a thermodynamically supercooled state. They can survive in a thermodynamically supercooled state because there are no nucleationsites,deepin the ocean.IT nucleated these fish would instantaneously freeze. Surface fish and plants, which have to survive in the presence nucleatingice crystals of in the sea water have developed a different mechanism of freeze avoidance.They use the antifreeze proteins,discussedearlier, to depress the freezing temperature their body fluids in a of non-colligative way (DeVries, 1971).Antifreeze proteins have the unusualability to depressthe freezingtemperature up to three ordersof by magnitude more than expectedfrom coUigative principles. It is not yet understoodhow this is accomplished. There are theories which claim that the antifreezeproteins depressthe freezing temperatureby binding to certain facets of ice crystals (Raymond and DeVries, 1977). In addition to their ability to depress noncolligatively the freezing temperature antifreeze proteins also have the ability to inhibit recrystalization once ice forms (Knight et at. 1984). Understanding how antifreeze proteins function may have many engineering applications. In particular, it could lead to the design of low temperaturerefrigeration cycles working with water. Recrystalizationinhibition hasalready found applicationsin the frozen food industry where it is desired to inhibit the recrysta1ization frozen foods. of Certain animals are freeze resistant The best known freeze resistant animal is the wood frog (RQJUlSylvatica) (Storey. 1997). Studies have shown that as the frog begins to freeze it produceslarge (half mol) quantities of glucose.The glucoseis producedin the liver and is distributed by the blood circulation to the remainder of the body. The frog can survive freezing to temperatures which up to 60% of in the body fluids are frozen. It cannot, however. survive freezing to lower temperatures.The freezing is extracellularand the cells in fact do not freeze, they only dehydrate.The purposeof

the glucose.is to decreasethe temperature at which 60 % of the body fluids are frozen (Storey et ai, 1992) In addition, the frogs produce ice nucleating proteins that are distributed in the blood vessels and ensurethat the freezing starts extracellularly(Storey,1997). TEMPERATURES HIGHER THAN mE PHYSIOLOGICAL TEMPERATURE OF WARM-BLOOD ORGANISMS. Researchtopics in bioheat transfer in the temperature range higher than the temperature of warm blooded animals deal primarily with three topics: high temperature organisms, of high temperaturein medicine use and accidents high temperature. The research at on high temperature organisms, (hyperthermophils) has become inportant in relation to the technique of PCR (polymerase chain reaction) {Huber and Stetter, 1998). The technique PeR useshigh temperatures melt of to the DNA and to producenew generationof DNA using enzymes from the high temperature organisms(usually hyperthermophilic bacteria). While the high temperature cycling of the PCR is not strictly a bioheat transfer problem, devices for PeR that do precise thermal cycling are of interest Also finding methods to precisely determine the temperatureat which the DNA strandmelts is an interesting problem of precise detectionof phasetransformation in very small samples with high temperature resolution. A good review of high temperature bioheatproblemscan be found in Diller (1992). In medicine elevated temperaturesare use to destroy undesirabletissues in a controlled way. During accidentsat elevated temperatures,the temperaturehas a similar effect on biological tissueslike in medical applications. except that here the application of heat is not controlled. Thereforein medicineit is important to precisely control the application of heat while in the study of accidents it is important to understand the relation betweentemperatureand the magnitude of damage. There are several methods for generatinghigh temperatures(hyperthermia) in tissue in medicine. They include: laser tissue interaction(Galletti. 1997; Cubedduet aI. 1997; Van HiUegersberg, 1997), focused ultrasound (Uchida, et aI. 1998; Albu Barkman, et aI. 1998), electrical inductance heating, contact heatingwith hot objects and electrical resistance heating. There are two areas of engineering interest in the study of high temperature biological heat transfer. The first area is of a

biological nature and relatesto understanding of the mechanisms of damage by elevated temperatures.The biological studies follow the pioneering work of Henriqueand Moritz (1947). They attempt to develop correlations between temperature,time of exposureand damagein an Arhenius type plot. The secondarea relates directly to heat transfer and deals with the propagationof heat into tissueand mathematical models of the heat transfer process (Diller, 1992). The strong interest in hyperthermic treatmentof malignant tumors has generated the renewed interest in developing models for bioheat transfer discussedin one of the earlier sections. CONCLUSION This article is, by necessity, very brief a introduction into the many topics of heattransfer in biomedical engineering and biotechnology. There is no doubt that this limited spacecannot do justice neither to all the topics in the field nor to many researchers that have contributedto the work in the filed. My hopeis that this paperwill serve as an introduction into the area of biological heat transfer to our colleaguesin the generalfield of heattransfer. RBFBREN CBS Albu Barkman, C., Kirkhom, T., Almquist, L0., Holmer, N-G., 1998,"Measurements the of thennal focus of an experimental focused ultrasound thermotherapysystem." International Journal of Hyperthermia,Vol. 14,pp. 383-393. Ananthanarayanan, V.S. 1989, "Antifreeze proteins: structural diversity and mechanismof action" Life CMm. Reprts.Vol. 7, pp. 1-32. ASHRAE Handbook of Fundamentals(1997) ASHRAE Press. Baish, J.W., Ayyaswamy, P.S., Foster, K.R., (1986), "Heat transportmechanisms vascular in tissues:a model comparson." of Biomech.Bog. J ViI. 108, pp. 324-31. Beattie, G M., Crowe, J H., Lopez,A D., Cirulli, V., Ricordi, C., Hayek, A., 1997,"Trehalose:A cryoprotectant lhat enhances recovery and preserves funclion of human pancreatic islets after long-term storage." Diabetes,Vol. 46, pp. 519-523.

Benedict, F.G., Talbot, F.B., 1915, "The physiologyOf the newborn infant, Characterand amount of katabolism", Carnegie lost. Wash. Publ. No. 233 Bischof, J., Rubinsky, B., 1993, "Microscale Heat and Mass Transfer of Vascular and IntracellularFreezing,"J. of Heat Transfer, VoL, 115,pp.I029-IO35. Carey, C., et aI. (Ed.). 1993 "Life in the cold: Ecological. physiological. and molecular mechanisms"., Ninth International Symposium on Living in the Cold, Fort Collins, Colorado, USA. 1993. Westview Press, Inc.: Boulder, Colorado, USA; Westview Press: Oxford, England,UK.. Chamy, c.K.,l992, "Mathematical models of bioheattransfer.in Bioengineeringheat transfer, Advancesin heat transfer, vol 22 (ed. Y.!. ato) pp 91- 155.AcademicPressBoston. Chato, I.C., 1980, " Heat transfer to blood vessels" of Biomed. Bog. Vol. 102, IIG-118. 1. Chato,J., C., 1981 "Reflections of the history of heat and mass transfer in bioengineemg- J. of Biomechanical Eng. Vol 103.pp 97-101. Chen, M.M., Holmes, K.R., 1980, "Microvascular contributions in tissue heat transfer", Annals of the New York: Academy of Science, Vol. 335, pp. 137-150. Cubeddu,R., Sow. C., Taroni, P., Valentini, G., Bottiroli, G., Croce, A C., 1997, "Study of mechanicaland thermal damage in brain tissue after ablation by erbium-Y AG laser." Lasers in Medical Science,Vol. 12,pp. 21-30. DeVries, A.L., 1971 "Glycoproteins as biological antifreeze agents in antarctic fishes", Vol. 172,pp 1152-4. Diller, K.R., 1992 "Modeling of bioheat transfer processes at high and low temperatures In Bioengineering heat transfer" in Advances in heattransfer" Vol. 22 (ed. Y.I. Cho) pp 91- 155. AcademicPressBoston. Diller, K R., 1997 "Engineering-based contributionsin cryobiology." Cryobiology, Vol. 34, pp. 304-314.

Doering, C., Ermenlroul, B., Oster, G., 1995, "Rotary DNA motors." Biophysical Journal, Vol.69, n.6, pp. 2256-2267.

pp. 8115-S116. ..

Orobnis, E z., Crowe, L M., Berger, T.,

Anchordoguy, T 1., Overstreet,1 W., Crowe, J H., 1993, "Cold Shock DamageIs Due To Lipid Phase Transitions in Cell Membranes A Demonstration Using Sperm as A Model", Journal of ExperimentalZoology, Vol. 265, 0.4, pp. 432-437. Duman, I.G., Wu, D.W., Xu, L., Tursman.,D., and others, 1991, " Adaptation of insects to subzero temperatures" Quart. Rev. of BioI. Vol. 66 pp 387-410. Eto, TK., RubinslcyB.,1996 "Bioheat Transfer" in Introduction to Bioengineering. SA Berger, W Goldsmith. ER Lewis Eds.Oxford Press. Elston, T C., Oster,G., 1997,"Proteinturbines1: The bacterial flagellar motor.", Biophysical Journal,Vol. 73, 0.2, pp. 703-721. Elston. T.. Wang, H.. Oster.G., 1998"Energy transductionin A TP synthase." Nature(London), Vol..391, n.6666,pp. 510-513. Fastovsky, D. E. and D. B. Weishampel.1996 "The evolution and extinction of the dinosaurs. xvii+46Op. Cambridge University Press: New York, New York, USA; Cambridge, England, UK. Florez-Duquet, M., McDonald, R B., 1998 "Cold-induced thermoregulation and biological aging." Physiological Reviews,Vol. 78. n.2, pp. 339-358. Fortcrre, P., 1996, "A hot topic: The origin of hyperthermophiles"Cell, Vo1.85, 0.6, pp. 789792. Galletti. G.. 1997. "Low power laser therapy: A noninvasive highly effective therapeutic modality." LaserTherapy.Vol. 9. pp. 131-136.

Hays, L M., Feeney, R E., Crowe, L M., Crowe, J H., Oliver, A E., 1996, "Antifreeze glycoproteins inhibit leakage from liposomes during thermotropicphasetransitions." Proc. of the Nat Acad. of Sciences(USA) Vol. 93, pp. 6835-6840. Hays,L M., Feeney, E., Tabfin. F., Oliver, A R E., Walker, N J., Crowe, L M., Crowe, I H., 1997, "FISh antifreeze glycoproteins protect cellular membranes during lipid-phase transitions." News in Physiological Sciences, Vol. 12,pp. 189-194. Henrique,F.C., Moritz, A.R., 1947, "Studies of thermal injury. I. the conduction of heat to and through skin and the temperatures attained therein. A theoretical and experimental investigation."Am.J. Path.Vol. 23, pp. 531-549. Holmer, I. Nilsson, H., 1995, "Heated mani1cins as a tool for evaluating clothing." Annals of Occupational Hygiene, Vol. 39, n.6, pp. 809818. Huber, H., Stetter, K. 0., 1998, " Hyperthermophiles and their possible potential in biotechnology." Journal of Biotechnology, Vol. 64, pp. 39-52. Ishiguro, H., Rubinsky B., 1994, "Mechanical interactionsbetweenice crystals and red blood cells during directional solidification" Cryobiology, Vol. 31, pp 483-500. Ishiguro, H., Rubinsky, B, 1998, "Influence of fish antifreeze proteins on the freezing of cell suspensions with cryoprotectant penetrating cells." lot J. of Heat and Mass Transfer, Vol. 41 pp. 1907-1915.
Kenney, W L., Havenith, G., 1993 "Thermal physiology of the elderly and handicapped: Heat stress and age: Skin blood flow and body temperature."Journal of Thennal Biology, Vol. 18,n.5-6, pp. 341-344.

Galtier,N., Tourasse,N., Gouy, M.. 1999. A

non -h yperthermoph iIi c common ancestor to extant life forms", Science,Yol. 283. pp. 220222. Hargreaves, M., Febbraio, M., 1998, "Limits to exercise perform,1nce in the heaL", International Journal of Sports Medicine, VoL 19, n.SUPPL. 2,

Kitamura, K., Tokunaga, M., Iwane, A., H., Yanagida. T., 1999, "A single myosin head moves along an acto filament with regular steps of 5.3 nanometers",ScienceVol. 397, pp. 129134.

Knight C.A. DcVries A. L., Ootman L., D., 1984, "Fish antifreeze protein and the freezing and crystalization of ice". Nature, Vol 308 pp 295-296. McGrath, 1.1., Diller, K.R., 1988, "Low temperature biotechnology: emerging applications and engineering contributions. lITD-Vo198, ASME, NY McGrath, ]J., 1997. "Quantitative measurement of cell membrane transport: Technology and applications", Cryobiology Vol. 34., pp. 315334. Mitchem, J.W., Myers, G.E., 1968, ..An analytical model of the countercurrent heat exchangephenomena", Biophys. J., Vol. 8, pp. 897-911. Negulescu, P.A, Rubinsky, B., Fletcher, G.L., Machen, T.E., 1992, "FISh Antifreeze Proteins Bloc~ Ca Entry into Mammalian Cells.."Am. J. Physiol.. 263 (Cell Physiol. 32): VoL 263. pp. C131O-C1313. O'Neal, 1., Rubinsky,B., and Phibbs,R.H., 1984 ..A Standard Experimental Procedurefor the Evaluation of Incubators," ASME Paper##84WAlC-8. Onik,G., Rubinsky, Bo,Watson, G., Ablin., R.I., 1994 "Percutaneous Prostate Cryoablation" , Quality Medical Publishing,Inc., St Louis MO. Pegg,D.E., Karow, A.M., 1987,"The biophysics of organ cryoprcscrvation" NATO ASI Series A: Life SciencesVol 147,PlenumPress, NY Pennes. H.K, 1948 ..Analysis of tissue and arterial blood temperature in the resting forearm." 1 of Applied Physiology, Vol. 1, pp. 93-122. Quinn, PJ., 1985, "A lipid phase separation model of low temperature damageto biological membranes,"Cryobiology, Vol. 22 pp 128-147. Randolph,S E., 1994"The relative timing of the origin of flight and endothermy:Evidencefrom the comparativebiology of birds and mammals. Zoological Journal or the Linnean Society, Yol. 112,n.3, pp. 389-397. Raymond, I.A., DeVries, A.L. 1977, "Adsorption inhibition as a mechanism of

freezing resistance in polar fishes" Proc. Nad.Acad.Sti. USA, Vol 74. pp. 2589-2593. Ring, R. A., Danks, H. V., 1998, "The role of " trehalose in cold-hardiness and desiccation. Cryo Letters,Vol. 19, pp. 275-282. Ruben, J., 1996, "Evolution of endothermy in manunals, birds and their ancestors., in " Johnston,I. A. and A. F. Bennett (Ed.). Society for Experimental Biology Seminar Series, 59. Animals and temperature: Phenotypic and evolutionaryadaptation;Meeting of the Society of ExperimentalBiology, St Andrews, Scotland, UK. xvi+419p. Cambridge University Press: Cambridge, England, UK; New Yark, New York. USA. pp.347-376. Rubinsky, B., Pegg, D.E., 1988, "A MathematicalModel for the Freezing Processin Biological TISSue,"Proc. of the Royal Society, Vol. 234, pp. 343-358. Rubinsky, B., 1989, "The Energy Equation for Freezing of Biological Tissue," I. of Heat Transfer, Vol. Ill, pp. 988-996. Rubinslcy.B.. Arav. A. Mattioli. M.. DeVries. AL.. 1990 "The Effect of Antifreeze Glycoproteinson Membrane Potential Olanges at Hypothermic Temperatures: Biochem. Biophys. Res.Comm.. Vol. 173.pp. 1369-1374 Rubinsky, B., Arav, A., Aetcher, G.L, 1991, Property of Antifreeze Proteins," Biochem. Biophys. Res.Comm., Vol. 180 No.2, pp. S66571. Rubinsky, B., Mattioli, M., Any, A, Barboni, B., Fletcher, G.L, , 1993 "Inhibition of ea2+ and K+ Currentsby Antifreeze Proteins."Am. J. PhysioL.(Reg. lnt Corp. Physiol.) Vol. 262. pp. R542-R565. Rubinsky., B., 1998 "Microscale heat transfer in biological systems at low temperatures". in Microscale Energy Transport, C-L Tien, A. Majumdar,F.M. Gerner,cds.Taylor & Francis. Saito, T., Sessler, D I., Fujita. K., Ooi, Y., Jeffrey, R., 1998, "Thermoregulatory effects of spinal and epidural anesthesiaduring cesarean delivery.", Regional Anesthesia and Pain Medicine, Vol. 23, n.4, pp. 418-423.

"Hypothermic Protection A Fundamental -

Shitzer, A. Eberhart, B.. (ed) 1986, "Heat transfer in medicine and biology; analysis and applications, Volland 2, Plenum Press,New York, 1985 Shock. N.W., aging in man." Ann. Rev. Physiol. Vol. 23 pp 97
1961, .. Physiological aspects of

for Fe(llI) reduction on early Earth", Nature (London),Vbl. 395, n.6697,pp. 65-67. Vogel, G., 1999, "RNA study suggests cool cradleof Hfe", Science, Vol. 283, pp. 155-156. Wang, J.C.H., 1982,"Hibernation and torpor in mammals birds." AcademicPress,NY. and Wang,H., Oster,G., 1998,"Energy transduction in the Fl motor of ATP synthase", Nature (London),Vol. .396,0.6708,279-282. Weinbaum,S., Jiji. LM., Lemons, D.E., 1984, "Theory and experiment for the effect of vascular microstructure on surface tissue heat transfer", J. of Biomed.Eng. Vol. 106,321-330. Weinbaum, S., 1iji, I.M., 1985, " A new simplified equation for the effect of blood flow on local average tisssue temperature", 1. of Biomech.Eng. Vol. 107,pp. 131-139. Wulf, W., 1974, " The energy conservation equation for living tissue", IEBE Trans. of Biomed.Eng. Vol. 21, pp494495S. Xu, X; Werner, I., 1997, "A dynamic model of the human~lothing-environment -system" , Applied Human Science, Vol. 16, n.2, pp. 61-75.

Siegel, D P., Green, W 1., Talmon. Y., 1994, "The mechanism of lamellar-to-inverted hexagonal phase transitions: A study using , temperature-jump cryo-electron microscopy" Vol. 66, n.2 pp. 402-414. Biophysicalloumal, Singer, Mike A., Lindquist, Susan., 1998, "Thennotolerance in Saccharomyces cerevisiae: The yin and yang of trehalose", Trends in Biotechnology, Yol. 16.pp. 460-468. Stoljwick. J.A.,J., Hardy, J.D., 1966, "Temperature regulation in man- A theoretical study" Pflugers Arch. Ges. Physiol., Vol. 291, pp. 129-162 Storey. K., B., Bischof, I., Rubinsky.B., 1992, "Cryomicroscopic Analysis of Freezingin Uvcr of Freeze-Tolerant Wood Frog," Am. 1. Physiol., Vol. 263 (Reg. Int. Compo Physiol., 32) pp. RI8S-RI94. Storey, K B., 1997,"Organic solutesin freezing tolerance." Comparative Biochemistry and Physiology A, Vol. 117,0.3, pp. 319-326. Tablin, F., Oliver, A E., Walker,N I., Crowe,L M., Crowe, 1 H., 1996. "Membrane phase transition of intact human platelets:Correlation with cold-induced activation", lournal of Cellular Physiology,Vol. 168.n.2, pp. 305-313. Uchida. To, Muramoto, Mo, Kyunou, H.. Iwamura. M., Egawa, So, Koshiba, 1(., 1998 "Qinical outcome of high-intensity focused ultrasound for treating benign prostatic hyperplasia: Preliminary report" Urology, Vol. ppo66-71. Vale. R.D.. 1993 "Measuring single protein motors at work", Science,Vol 260. pp. 169-170. Van Hillegersberg. R., 1997, "Fundamentals of laser surgery." European Journal of Surgery, Vol. 163, pp. 3-12. Vargas, Mo, Kashcli. K., Blunt-Harris, E., L, Lovley, D., R., 1998."Microbiological evidence