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History of biology related to low temperatures.
( Edited by Dr. V. Belgrano and Dr. E. Nieddu ).


In these chapters we will discuss the physical approach to the freezing of biological tissues, starting, of course, from their fundamental element: the cell.
We will study the fundamental principles of physics that studies started in the 60s, will then be adopted ( and implemented ) until you reach the modern cryosurgery.
It is not our task to describe all developments "historical-scientific" and mention of the scientists who have contributed to this progress, but we will discuss the historical evolution of cryogenics in its essential steps discussing the key developments and biophysical implications that, today, are the basis of cryosurgery.
We cannot forget, however, a scientist who has spent his entire life in search of  the biological laws relating to the cold.
We refer to Peter Mazur distinguished biologist and  fruitful pioneer.
We have no other way to do honor to this scholar who to describe a brief autobiography, however, obtained directly from the documents that we have found on the net and from qualified scientific libraries of the various Italian universities and the world.
In continuation of this series we will discuss other fundamental scholars with apologies to those who we did not mention, we proposing to speak later of their important and useful work.

Peter Mazur

Research Statement

My field is cryobiology, the branch of biology that is concerned with the responses of living cells to freezing and very low temperatures. All living cells require liquid water and appropriate temperatures to function. Freezing constitutes a dramatic perturbation of these conditions, and consequently, the study of how cells respond to this perturbation may help elucidate the role of liquid water in living cells and in intracellular constituents such as proteins and nucleic acids. Furthermore, these responses involve fundamental physiological and physical chemical processes like osmotic flow; and freezing injury primarily involves damage to the cell’s outer membrane, a vital and complex component of cells.
For the past eight years, our laboratory has been analyzing the involvement of these physical processes and forces in the survival or death of cryopreserved unfertilized eggs from mice, zebrafish, and the frog Xenopus, and cryopreserved yeast, and Chinese

Hamster V79 and COS-7 tissue culture cells. We chose these cells primarily because they serve as excellent models for examining the mechanistic aspects of the field. But simultaneously much of cryobiology has had and will continue to have major practical ramifications. In 1972, my group, then at the Oak Ridge National Laboratory, became the first to successfully cryopreserve early mouse embryos. This led in rather quick succession to the successful preservation by others of cattle and then human embryos. The first has played a major role in the genetic improvement of livestock; the latter has played a major role in clinical assisted reproduction.
One intriguing aspect of cryobiology is its relevance to the search for extraterrestrial life. In this venture, NASA has made its goal "To follow the trail of liquid water," Mars is tantalizing in this regard. It is the current consensus that long-long ago, the planet had considerable liquid water but that it was subsequently lost because of decreased temperature and atmospheric pressure. But perhaps not all has been lost, for the recent surface rovers have obtained photographic evidence of its existence.
(original version)

These are his words, simple and clear, which reveal the greatness of a man who for us, scholars of cryogenics, we believe one of the fathers of the low temperatures.
Through his  past and present studies it was possible to understand the complex laws that govern this field charming and articulate.
Recall that Dr. Mazur received his AB magna cum laude from Harvard University in 1949 and PhD in Biology from Harvard in 1953.
After four years with the Air Force Research and Development Command, he spent two years at Princeton University as   post-doctoral fellow National Science Foundation before joining the staff of the Division of Biology in 1959. In addition to   of these fundamental aspects of cryobiology, Dr. Mazur is dedicated to applications of cryobiology in medicine, agriculture, and genetics. For example, his laboratory was the first to freeze the embryos of mammals:  discovery that has had a significant impact in all three areas.

In 1972, the biologist Peter Mazur and two colleagues have announced a startling discovery: they had frozen the  embryos of mouse, holding them for days in liquid nitrogen ( -196 degrees Celsius). Once thawed were implanted into a surrogate mother.
The cover of Science magazine showed the results of the experiment : mouse embryos frozen, frost-free.
In two weeks of testing, the team had figured out what the others cryobiologists (literally, "biologists of the frost" ) looked  for for two decades.
The technique was soon adopted by the industry of cattle: the frozen embryos are now routinely in the United States.
The freezing of embryos has not received the deserved success until 1980, when he made his entrance into the world of fertility clinic man.
Despite the concerns raised by human embryonic freezing, Mazur is realistic about the future use of cryobiology: the defense of animals whose species is endangered, for example, the implantation of embryos from horses with rare zebras and maintain mice for research or of fruit flies, protected from, say, the "fire" of laboratory or studio straightening of mutations called "genetic drift".
Eventually, the cold technique could also be adapted to preserve human organs for transplantation-related, while their guest is gradually introduced to his new tissues. Two decades after he made headlines with the technique of cold, Mazur still has a special place in our hearts for his important work done.
It  was appointed a member of the United Nations in 1985.

It 'also interesting to note how this biologist in his writings never tires of stressing the importance of thermodynamics and physical chemistry. Basic sciences for understanding and cryogenic studies, as already stated above, for the correct design of the  machines for the cryosurgery. The effects of the animal tissues subjected to low temperatures are not yet well known. Only the cryobiology can help to better understand them, favoring, consequently, the development of equipment for cryogenic interventions  ever more effective and safe.
At present, the main knowledges dated back to studies conducted in the last decades of the century 1800, this researches, which had already been described and had clarified in detail the main mechanisms, they left quite a few unsolved problems of biophysics.
In particular, research conducted on the "preservation" and "conservation" of the tissues by the action of cold, have proved to be of extreme importance for the understanding of the mechanisms of "destruction"  of tissue, for therapeutic purposes (which, in actually, it is our fundamental objective).


Freezing point .

The cell, from the structural point of view, is composed of a cytoplasm ( enclosed by a plasma membrane) in the interior of which there is a core ( contained in a nuclear membrane); under the chemical aspect is constituted by a set of proteins, carbohydrates, lipids, ions and water whose exchanges with the outside world are selectively regulated by pores, protein channels and carriers present on the cell membrane. Consider, now, the case of a lowering of the temperature to which the cells are subjected in an aqueous suspension.
For one thing happens freezing of water in which they are immersed; later, with the gradual decrease of temperature,  the ionic solution of the cytoplasm , which has a freezing point of between -6°C and -10°C, supercool until reaching a temperature such as to also freeze the intracellular water [ 1, 2 ]. From experimental observations it is noted that the water within the cell freezes spontaneously only after it is formed the external ice [3]. This phenomenon is caused by a combination of factors that will be analyzed later to better understand their role in cryobiology.

Crystallization .

The freezing of the water, as occurs in the process of creation of the crystals, requires initiators. We can therefore say that the ice has the effect of external "trigger" on the crystallization of the intracellular water.

Surface tension.

A further important parameter which intervenes in the freezing phase is represented by the surface tension. All molecules that are in a system will attract ( or repel ) mutually second forces, said intermolecular, the origin of which can be caused, in addition to a number of other factors, by bond hydrophobic, electrostatic effects or by the presence of bond  forces of Van der Waals. The main parameter of the forces is formed by the distance between the molecules themselves. This explains that, when a molecule is located inside a liquid, has a well defined potential energy due to the contribution of particles that surround it, and when, instead, is placed on the surface, is subject to a strong attraction to the fluid ( and there component is no attraction towards the outside gas or vapor). It follows that, if they are placed on the surface of all the molecules are attracted towards the inside, the configuration of minimum energy of the surface will have to be necessarily in the form of a spherical drop.
In the case in which the attraction of the liquid molecules to the solid is weak, there will be a deformation of the drop due only to the effects of gravity. You will get a slight deformation of the sphericity. In contrast, when the solid exerts a strong attraction, the deformation of the drop will increase until the complete shedding of the liquid on the surface of the solid (a phenomenon known by the term of wettability of the surface).
It is possible, at this point, calculate the work (infinitesimal increment) which allows to define the energy needed to increase the surface area of 1 cm 2, and that is, the force along 1 cm multiplied by the distance of 1 cm within which said force moves.

When the water comes out from the cell, within this increases the concentration of solute and, consequently, the freezing point decreases.
We know that the freezing  point (and the vapor pressure) of a solution is inversely proportional to the concentration of solute. This phenomenon is determined by a number of factors such as: the cooling rate, the membrane permeability to water and solutes, the temperature coefficient, the initial volume of water and the number of moles of solute in the osmotic cell [4].
From thermodynamic considerations that bind analytically the parameters outlined above, through the use of numerical solutions, was represented graphically the percentage of initial water that remains in a cell at various temperatures for different cooling rates. Experimentally, it has been noticed that the cooling obtained in a short time, prevents the water from completely exit from the cell, and the greater is the water inside the cell, the greater the supercooling.
The phenomenon   is reversed   when   the cooling rate   is lower.
In   first approximation , we can assume  the shape   of the cell   to that   of a sphere   and ,  since the ratio   between volume and   surface is  
r3  /  r2,  we could assert   that ,  at   the same   cooling rate,   the   larger   cells   detain   a higher   amount of water   inside.   This explains the   real   difficulties encountered   in being able to   successfully freeze   cells   very large   without getting any   physical impact   on them   [4].



As we have seen ,  the lower the   cooling rate,   the greater is the   removal of water   from the cells.   This fact   is useful   to prevent freezing   inside.   Of course ,  the elimination of water   from the cell   has as a consequence   the increase   of the concentration   of the solute .  Of chemical injury   that follows ,  the   NaCl   is   considered to be the   most responsible .  At the   present state of our   knowledge   of this damage , meaning  break   or   change   of chemical bonds   between   elements   due to   physical effects   on the system , we know little.
However, we can   affirm that , the higher the  thermal gradient  and   the greater will be   the damage   done.

We are therefore   in the presence   of a   partial   favorable effect   namely:   a slow   cooling rate   prevents   the physical damage   produced by the   ice   inside the   cell, but   is not   able to reduce   chemical damage   that ensues.
We can say, moreover, that, complete dehydration for the lowering of temperature, further decreases in temperature can be made at any speed without causing any damage to the cell.
The heating takes place respecting the same rules that have been set forth for the cooling and the water that was previously spill, come in  into the cell at a speed that is a function of time, of the same temperature and concentration. Also in this situation, it has been shown experimentally that there is a different cell damage at different speeds of thawing. Normally it is observed that,  more rapid heating, the greater the survival of the cells [5, 6, 7].


In the 1970s the theoretical knowledge relating to the use of cold in surgery began to take consistency thanks to scientific researchers do not typically Cryobiologists but also doctors and surgeons assisted by engineers and physicists. The theoretical and experimental work of Prof. Mazur remained as an important and undisputed reference .
The modern cryosurgery search the deleterious effects of cold at the end destructive.
And it is this aspect that is relevant for the design of the tools and techniques of Cryosurgery.
The maximum destructive effect of a cryogenic instrument, in accordance to what has been stated above and in accordance with the research of Prof. Mazur, is to work on a given tissue by freezing it very quickly and then heat it slowly.
Unfortunately, a tumor mass cannot be represented by a simple mathematical formula or a regular geometric shape.
In essence it is a model "real" and must, therefore, take into account three key variables: 1) the accessibility of the area to be treated, 2) the characteristics of the "mass" to be cooled, 3) any cold damage to the perilesional tissues.  
When it comes cooling  in the unit of time, it inevitably we must reference to the  cryogenic power. To cool quickly determined masses of tissues it is requiring high power, of course, much greater than that which would necessarily be used in a normal cooling (long times).
In this "historical" period of cryosurgery the procedures are not yet well defined. The protocols that are able to determine and follow the cryogenic action in its complex are in the early stages.
Also the medicine began to propose process chemicals to solve problems that cryosurgery was not able to resolved.
And just about the evolution pharmaceutical we propose a brief overview to describe how in the world is produced in this regard and in particular the study of the complex phenomenon related to different aspects of the immune response when working with cryosurgery [8-12 ].

Immunological response .

The immunological response to the freezing has been the subject of detailed examination by Sabel.
Shulman, Ablin et al. demonstrated that specific antibodies appeared in the serum of rabbits after freezing of the prostate. It was stated that the rupture of the membranes of the cells due to the freezing should release sufficient antigen to induce the formation of antibodies.
The interest of the immunological response was greatly enhanced in 1970 when Soanes et al. described the reduction of metastatic lesions as a result of repeated freezing in patients with prostate cancer.
Many researchers have discovered the benefits of the specific action on the immune system by stimulating the production of antitumor antibodies, such as T cells and cytokine production. Some scientists have thought that the immune response could be increased with adjuvant agents.
Neel and Ritts reported that the increase of the specific immune response of the tumor, seen after freezing, increased with excision of the tumor, twenty four hours after freezing. It was thus suggested that the majority of the immune response that follows the tumor necrosis caused by freezing happen in the first twenty-four hours.
Seifert et al.  by freezing 50% of the liver of rats, they noticed an increase in serum levels of cytokines, causing an injury to the liver and kidney, probably mediated by the release of cytokines from the liver itself. Joosten and his collaborators described a high level of serum cytokines after freezing of tumors implanted in mice.
In conclusion, these studies reflect the diversity of opinion about the benefits of the response of the immune system in cryosurgery. Although experiments with animal tumors demonstrating growth control through the freezing techniques, clinical experience has shown benefits in cryosurgery in human cancers that could be attributed to the immunological response. On the other hand, it is clear that the larger the volume of tissue destroyed by freezing, the higher the likelihood of liver dysfunction and postoperative lung (CRIOSHOCK), which was attributed to the release of cytokines and products related to the break of tissue; as has been demonstrated in some animal experiments the response of the immune system, are needed subsequent studies on cryoimmunology.
In general, there is a reduction of replication and transcription of DNA, a slowdown in protein synthesis, except for the increase of synthesis of particular classes of proteins, the shock proteins, proteins induced by the shock. Surprisingly these proteins are produced not only by the thermal shock, but also by other agents stressful for the cell (alterations in pH, pressure, tonicity etc..).
The high conservation and inducibility suggest that even heterologous shock proteins can be used to treat human diseases.
The importance of these shock proteins is increased by the fact that, in addition to a role as intracellular protecting the integrity of the cell, they retain an extracellular important role in immunity.
The shock proteins are important factors involved in the development, oncogenesis, neurodegenerative diseases and autoimmune diseases, viral infections and old age. Why?
For example, cellular senescence and aging in vivo would be correlated with a continuous decline in the ability to produce these proteins in response to stress.
It is precisely the role of the extracellular who is involved in the immune system: increased serum levels of shock proteins in humans are recorded as a response to stress conditions, including inflammation and viral and bacterial infections. Stress and the increase the release of proinflammatory cytokines shock proteins from tumor cells.  [Nieddu E. et al, 2013 CPD awaiting publication].
The previous report suggests that the reactive effects of the immune system when the subject is subjected to cryosurgery provides a plausible and elegant theory that Dr. E. Nieddu made about the consequences (positive) of the cold.
This theory explains that in general the biochemical processes due to the effects of the low temperatures will be, in our opinion, very important to the future consequences of the fight against cancer. And, very importantly, provides other scientific justification to the choice of the interventions of cryosurgery in all cases of risk tumors and also in those that can operate with traditional techniques.
In other words this research, it is  far from to be completed. The future results will lead  to potential solutions that will certainly be a positive turning point in the fight towards the treatment of cancer using low temperatures.
The development of cryosurgery as a therapeutic technique has received a greater stimulus by the introduction of automated equipment  cooled with liquid nitrogen (-196°C) thanks to Cooper and Dr. Ing. AS Lee in 1961 [13].
In his autobiography, Cooper, neurologist in New York City, described the details of the development of a probe cooled with liquid nitrogen under vacuum for act on the brain and the contribution of engineers and scientists from the Linde Division of the Union Carbide Corporation for the construction of apparatus [13]. This instrument was consisted of a isolated rod that leads    a metal tip . The apparatus is able to control  the temperature and had the ability to adjust the thermal effect on the probe.
When applied to tissues, it was able to extract heat with continuity and  produce a cooling  in situ. The device was designed to produce cryogenic lesions in the brain for treatment of patients
parkinsonian and other neuromuscular disorders [13]. Cooper acknowledged that the instrument had a utility in the treatment of tumors. He interested his fellow experts in other fields of clinical techniques of the cold, and from that moment began to be called cryogenic surgery or cryosurgery.
This tool has become essentially the prototype from which all future probes criochirurgiche using liquid nitrogen have been developed.
The work and research of Dr. I. Cooper in the field of cryosurgery neurological gave an indelible mark in the field of international scientific and the consequential neurosurgical applications have led the researchers to be in agreement that the 1960s represents the beginning of the modern era of cryosurgery. We also allow us to state that in recent years also began the modern era of cryobiology (thanks to the  studies and discoveries of Professor P. Mazur).
The probe was designed by Cooper and Lee AS is not a simple (but effective) mean of surgery but a "keystone" for future applications. This apparatus paves the way permanently to new surgical and biological methods.
It is therefore only right to renew our thanks to these enlightened and  unforgettable  scientists.



From the years of its appearance, thanks to the studies of Professor. Cooper and Eng. Lee, the modern cryosurgery has never stopped in its evolution. In 1993 we verify its rebirth in particular in the treatment of localized prostate cancers. The methods described by Onik et all. have been adopted by most of the cryosurgeons[14].
The attempt was the complete destruction of the gland but that you could not realize [22]. You could get an ablation of cancer from 80% to 90% of interventions known [17-20].
Greater the smaller tumor size were the positive results [26,28,30]. This fact is explained by checking the spread of the cold front and its thermal distribution. One must remember that the destruction of a biological tissue occurs at certain temperatures well below which will get different inflammatory effects but not the killing of the cells.
In 1994, the use of five probes was considered a best practice in view of the fact that the contribution of temperature sensors in the information posed a better perspective of surgical intervention [31] [26].
The use of eight cryoprobes was certainly an improvement in the destruction of tumors and the positive effects that  we verify on the patients.
It is as presented  by prof. Fred Lee comparing a group of patients treated with 6 - 8 probes with the one previously treated with the method to five probes. The reference points were the berries epithelial residues (REA) and serum prostate-specific antigen (PSA) obtained six months after the cryosurgery [26].

COMMENTS   Prof   Lee.

The comments expressed in this research by Professor Lee announce wide-ranging vision and, let's face it, futuristic interventions of the cryosurgery.
These assertions will find in the following pages and they are the confirmation of our "experimental" observations. This give us a little hope for a successful continuation of that science.
Cryosurgery continues to be driven by biological research and technology. A system to eight cryoprobes and knowledge of absolute killing at -40°C facilitates our search for the complete ablation of the prostate [32]. The objective sought by the researcher is to get closer to a complete destruction of the cancer of prostate A system to eight cryoprobes and knowledge of absolute killing at -40°C facilitates our search for the complete ablation of the prostate [32] gland. The use of 8 probes has increased by 3.5 times compared to the conventional method of the five probes and identifies 89% of our cases.
The previous   use   of the five   cryoprobes   produces   pathological results   comparable with those   of the other   by   radiation   therapy   [ 33, 34].
The   concluding words   of Professor   Lee   are   the best hope   future: "Our approach  would help   standardize   cryosurgery   and produce   more   easily   a framework of experience .  A perspective of   multi- institutional  studies   could be set up".


Laboratory tests and   physical   mathematical   simulations .

The previous work of prof. Fred Lee and co-workers is, in our opinion, an indicative step that confirms our discussion on the progress of the surgery of the cold.
We are mindful  these observations may, in part, and in light of the present findings, be questionable, nothing prevents to think that a greater number of probes can certainly favor the cryosurgical intervention.
In our humble opinion, however, the critical observation must be focused not only on increasing the number of probes, but also in the methodology of the intervention and the reasons of physical-mathematical transfer  of heat  that are at the basis of these interventions.
The work of professors Baust and Gage at the University of Buffalo (NY), which was published in 2005, under the title "The molecular basis of cryosurgery" help us.
In this report, the authors propose that the modern molecular research has the potential to spread substantially the effectiveness of cryosurgery, especially in the treatment of tumors.
According to their experience, the critical aspect of this research is the recognition that apoptosis is a mechanism of cell death due to the effect cryogenic. This feature recently defined of  damage by freezing can provide an opportunity to manipulate the evolution of damage and to effectively manage the protection of healthy tissue by creating effective shelters so as to be useful in therapy.
In their article is considered the basis of biological cryosurgery and defines the technical practices that should increase the effectiveness of cryosurgical interventions. The lack of blood supply deprives the cells of each exchange for survival.
The importance of this damage mechanism, having as its characteristic the  vascular stasis in the tissue thawed, has long been analyzed, including recent experiments [35]. Nevertheless, from the molecular point of view, the most important step forward in basic research concerning the cryosurgery is the recognition that apoptosis is a mechanism of cell death that follows the damage by cold [36].
Apoptotic cells are characterized by a cut in the DNA non-random, by protrusions of the membranes, by the inversion of the phospholipids in the same and by the activation of caspase [38].
Apoptotic cells in the human colon carcinoma were discovered by Hanai et al. [15] after exposure to temperatures of bland freezing (-6°C to -36°C). Kinetic studies showed that the apoptotic cells could enter in this state for up to eight hours after heating; similar characteristics are reported by Yang et al. [16]. The relatively recent discovery of apoptosis in cells adds a new dimension to the possible therapy [17].
The assertion that cancer cells in many organs such as the prostate gland, kidney and liver, which require some specific techniques are used to ensure the destruction, it means having the scientific sensitivity towards the bio-instrumental techniques that can destroy the cancer in various cells using different procedures between the different natures of cancer.
The use of multiple probes allows you to get to the formation of more uniform ablation and low temperatures in the target tissue. The dimensions of the tissue to be cooled include the tumor and a slice of appropriate surrounding healthy cells, a comparable volume to the size of tissue to be cut off if the surgery was excision therapy choice. In the liver or kidney, 1 cm margins around the tumor are easily accessible, but the prostate gland size and   anatomical relationships  not permit these margins.
It must therefore have recourse to a technique which must be varied by using more cycles of freeze-thawing. In this way, it faces a new method that applies several thermal cycles by increasing the cellular destruction due by cryosurgical intervention.
The commonly accepted method long taught about the cryoablativ surgery  for tumors is to freeze the tissue so rapidly as to reach a temperature of cell death in the tissue. To freeze, then, up to a safe margin beyond the end of the tumor (without trespassing beyond the acceptable in healthy tissue), heat gently, and then repeat the cycle freeze-thawing as previously verified.
In this way one begins to speak of "time" and "cycles" of cooling.
The rate of cooling, heating and the temperature profile, vary with the distance from the cold probe.
The intracellular ice occurs within a wide range of temperatures, i.e. from 20-50°C / min. [19,20]. In  packed cells a cooling rate lower can also produce intracellular ice  [38].
The temperature produced in the tissue is critically important;    a tissue temperature from -40°C to -0°C should be produced in the tumor, within a safety distance around the tumor [39, 35, 20].
Some experimental data suggest that a tissue temperature of -20°C is appropriate for the tissue destruction but this recommendation should be viewed with skepticism. Certainly an extended tissue damage it occurs from -20°C to -30°C, but the cellular destruction is uncertain or incomplete.
When the tissue is maintained from -15°C to -40°C, as occurs especially in the case of the periphery of the frozen bodies, the biochemical changes and the growth of the ice crystals are increased, while also increasing the speed of cell death. However, the optimum duration of freezing is not well defined and may be specific to the cells and tissue.
The longer the duration of the thawing,   wider is the damage to the cells to the effects of the solute, the recrystallization of ice crystals, the prolonged oxidative stress and the growth of ice crystals.
The large ice crystals that form during the recrystallization "to hot" create  forces of  shear that break the tissues. This type of crystal growth is maximum from -15°C to -40°C, and especially from -20°C to -25°C.  A quick thawing increases the chance of cell survival, which is known for some time in the treatment of frostbite.
The repetition of the freeze-thaw cycle produces a cooling   faster and more extensive, so that the volume of frozen tissue is enlarged and the boundaries of certain  destruction of the tissue are moved closer to the outer boundary of the volume frozen. The first cycle freeze-thaw increases the thermal conductivity of the tissue caused by cellular destruction. The second cycle subjecting the fabric to physicochemical changes extended, while founded thermal damage for the second time. With repetition, the second cycle increases the area of necrosis in probably 80% of the volume previously frozen. The repetition is of special importance in the prostatic cryosurgery because the second cycle probably shifts the lethal isotherm close to -20°C, thereby allowing a closer approach to the margins of the gland without damaging the rectum. For this reason, the repetition cycle of freeze-thawing, critical in the treatment of all cancers, it is especially important in treating prostate cancer.
And it is in the year 2009 which is published a study of the process of cooling and thawing of a biological system by work KJChua and SK Chou Department of Mechanical Engineering, University of Singapore Nactional [39].
This research paves the way for a combined methodology between the various fields of science such as, biology, physics, mathematics, medicine and engineering.
And we believe that this approach interdisciplinary is very important for the results which were, however, very significant for the purposes of our investigation and of great importance and scientific instrument.
The researchers propose to be able to control the amplitude of the thaw and cooling within certain critical intervals of temperature in order to adjust the spatial extent of the destructive effects during cooling.
In order to view the isotherms in a specific area of specific interest was created a physical-mathematical model, through which operators are able to assess the damage in the tissues of specific interest.
The system has been tested and compared with data obtained experimentally by subjecting   samples of porcine liver to cooling.
One of the advantages of cryosurgery is the possibility of locating the treatment, also mathematically simulating the cold front and thus minimize damage to surrounding tissues. The doctors K.J. Chua and S.K. Chou show that in the cryosurgery are not present in most  of  effects of post-operative characteristic of conventional interventions of cancer.
They point out, moreover, that the cold does not guarantee the complete destruction of tissues. Actually, to apply this technique, it is essential to determine the thermal parameters that cause the destruction  of actual tissue [39].
In this research has been introduced the term "model" understood in the broadest and extensive sense of the term.
A mathematical model of experimentation calibrated, therefore, becomes an effective tool in the planning of cooling and thawing protocols. Despite the actual extirpation of the tumor obtained by cryosurgery, the basic cooling-thaw cycles, if not reasonably improved, they cannot produce results as the ones you want. The optimal technique of cryosurgery, therefore, requires a thorough understanding of the cooling-thaw cycle with its biological components.
Becomes essential to create a complete and extensive physical schema-biological-mathematical able to promote and represent the biophysical changes which occur in the tissues during the surgery of cryosurgery. This model must incorporate, and thus to represent multiple stages of physiological changes and, therefore, critical and complex when considering the various biological processes involved.

In the study from the University of Singapore has been developed, however, a model to study  thermic important operating parameters such as the number of cooling-thaw cycles, as the temperatures of melting and cooling intervals between fronts during the freezing of a biological system. Therefore assumes a fundamental importance the ability to examine the degree of cell destruction by a mechanistic point of view, with respect to the amplitude of the cold and the degree of mobility cell.
And, remarkable fact, the model is able to draw the cold front in the range of (0°C and -50°C), it is really modern and useful for studies of the relationship between cooling-thaw cycles and the interval front of the sink.
The research was then undertaken with a broad vision, incorporating all the important analysis inherent in a model of heat transfer in a biological system.
Recent experiments have indicated a correlation between the speed of the cold front and the interval between its propagation and the boundary cell death [40,41]. This range can be used as a parameter to evaluate the effectiveness of each protocol of cryosurgical. To value  destruction of cancer cells in biological organs. When the range of the cold front is very broad, towards the end of cryosurgical intervention, the effectiveness of this operation may be compromised if the area of cell death is considered small, while a large region undergoes intra-and extracellular ice formation. In this situation, the surgeon needs to be warned against the under treatment. Ideally, the contour of the total cell death (-50°C  cold front) could approach the edge of the tissue, while the 0°C would reside in the 10 mm from the peripheral edge of the tissue itself. Since the development of the ice-ball is dependent on many operating parameters, such as the size and type of a cryoprobe, the value and efficiency of the cryogenic transport, the efficiency of heat transfer to the probes,  the thermal conductivity, specific and latent heat, and the distance between the place of the target and the probe, is more likely to get a range of a cold front more extensive than that of the ideal case [41].
Taking our the concluding observations of the two researchers, they remember that in order to obtain a desired level of cellular destruction in any biological system is essential   predict the temperature distribution, the profile and the position of the cold fronts in the biological tissue under examination.
We believe that this model is useful for clinical studies of cryosurgery as a valuable reference for new insights in the understanding of cryoablation of the diseased tissue.
It is to the end of the first decade occurring major scientific efforts to improve the cryosurgical techniques.
Improve means to understand the phenomenon and, in accordance with scientific laws, apply it or use it for the purposes of surgical procedures.
In this regard, the year 2009 was published a work of John G. Baust, Andrew A. Gage, Anthony T. Robilottto and John M. Baust which suggested, in fact, the responses of thermal therapies of the prostate cancer with a particular emphasis to the cryotherapic techniques  [43].

Recent findings .

Long-term studies clearly demonstrate the effect of the use of modern techniques of cryotherapy in the treatment of prostate cancer. The American Urology Association Best Practice Guidelines identifies prostate cryoablation therapy as both primary and conservative. Modern studies show and confirm the effectiveness of exposure at -40°C for lethal genotypes of prostate cancer with the technique of double freeze-thaw. It is reported, also, the use of additives to sensitize the cancer at low temperatures.
We see that the lethal values are around -40°C  not are very far from (-50°C) proposed by bioengineers at the University of Singapore.
The thermal therapies, especially cryoablation, are of increasing interest for the treatment of prostate and renal cancers. The methods for the application of cryogenic therapy and the mechanism of cell death underlying the freeze thaw cycle are clarified. The research focused on the development of sensitizing agents for the freezing is of central interest to promote the effectiveness of this therapy.



Despite the double freezing, some cells may still survive: it is therefore necessary the presence of additives: radiotherapy (cells cooled, in fact, show greater sensitivity to radiation) [43]; immunological  potentiators (freezing the cancer leads, in fact, to a immunological response with remission even metastatic), cytotoxic drugs, most commonly used approach (it is proved that the greater effectiveness is achieved when the drug is given 1-4 days before surgery) [44,45].
In recent years efforts, almost universal, of researchers in the world with regard to cryosurgery are oriented to the understanding of biophysical phenomenon. About published works are more refined and consistent image of the borders are reached.
Therefore, we are on the threshold of new events Cryosurgical that only a few years ago were simply unthinkable.
The publication of A.A. Gage, J.M. Baust and J.G. Baust: "Experimental investigations of cryosurgery in vivo" [46] confirms what we have just mentioned.
This is a rundown of operations of Cryosurgical made on all human organs. Describes the difficulties and successes.
This review provides a comprehensive overview of in vivo testing, which was the basis of the progress of this specific therapy. The effects on tissues and cellular events related to them. These phenomena that are the basis of the mechanisms of destruction are completely explored. Are highlighted the direct injuries on the cells, vascular stasis, apoptosis and necrosis; are presented, in addition, certain standards optimal of the freezing techniques to achieve more and more an effective therapy. The authors describe in vivo experiments on major organs, including wound healing, immunological aspects, the reason of the consequent response to thaw, and the use of strategies to increase the sensitivity of cancer cells to the effects of frost.

Direct   cellular injury .

The reference point for the direct cellular destruction by freezing is the formation of ice crystals that removes water from the cells and leads to a cascade of harmful events[47,48]. Recent in vitro investigations have identified apoptosis as a direct mechanism of injury [49]. This was confirmed by in vivo experiments (Steinbach and others), which show the presence of necrosis in the central part of the lesion, and apoptosis in the device, at 8-12 hours after the first application of cold [50]. Forest, injecting human pulmonary adenocarcinoma cells in mice, showed that the central part of the lesion was frozen necrotic, but the peripheral area had apoptotic cells. Apoptosis is progressively increased in a range of 2-8 hours after freezing, although the boundaries between the two were not clear. A second peak of necrosis was observed after four days [51,52].
Wen, by injecting cells of adenocarcinoma of the lung in the subcutis of the mice,  observed freezing in the central area of the tumor after necrosis. He observed an increase of 68% of the volume in four days. In the peripheral area of the lesion, has noticed an increase of apoptotic cells, with a peak 8-16 hours after freezing. It is thought that the apoptosis is due to mitochondrial damage and an increased expression of Bax protein and the activation of caspases [53]. These experiments confirm the importance of apoptosis in the cell death of the lesions, in particular in the peripheral region, at the same time allows us to highlight the close connection that exists between the experiments reported in vitro with those in vivo.

The vascular damage.

In recent years, modern cryosurgery has performed several experiments on fabrics of various kinds, such as rat liver, pig skin, rabbit, hamster.
All of these have shown damage to the microcirculation. During thawing, the frozen tissue before, shows progressively edematous and congested.
The cause of the edema is the damage of the junctions of the endothelial cells, already evident in the first two hours after thawing phase, resulting in increased capillary permeability, edema, platelet aggregation, and finally, thrombosis.
Most of the cells goes into necrosis at a temperature between  -20°C and zero degrees, the remaining in apoptosis. The relative importance of direct cell damage and microcirculation remains unclear, although both are important elements in the cryosurgical technique [54].


Technology - Methods  and Control   of freezing.

A wide variety of agents and cryogenic freezing techniques with different equipment was used in the experiments of cryosurgery. Scholars in the first half of 1900 realized by the freezing injury in different tissues and had gained considerable experience in the field of cryosurgery. These experiments have helped to pave the way for the modern era, which began (as previously mentioned) by Cooper and Lee in 1961 when they created automatic equipment cooling with liquid nitrogen (-196°C). Years later, the development of devices which cool more probes simultaneously or in sequence have improved the versatility of the techniques [ 55,56 ].
In recent years, argon (-186°C) and nitrogen oxide ( -89.5°C ), which had a limited use in the past, have become commonly used as compressed gases. They was used in instruments which utilized the effect which takes the name of the physicists who discovered it  Joule -Thomson (JT). The compressed gases, especially argon and nitrous oxide, have allowed the construction of various instruments, such as thin needle-like probes, probes catheter balloons structures, and clamps.
These modern devices, they had some differences in the ability to freeze mainly related to the type of cryogenic use. The volume of frozen tissue into a single application of a cryoprobe is directly related to the temperature of the probe, the area and duration of contact with the tissue. Another important factor is the thermal conductivity of the cell structure. To use two or more probes at the same time we introduce a measure of uncertainty in the provision in question is likely to create an entity of interactions during freezing. Everything is coordinated by special software [57].
Numerous studies have focused on monitoring the progression of freezing in the various tissues. They have been used needles special  thermocouples to measure the temperature, but these have not provided an overview of the volume frozen. Ultrasound imaging techniques implemented by Onik in 1980, allowed accurate monitoring in the freezing process [58,59].  In the  1990 it has been a renewed interest in cryosurgery. Ultrasound is the most common method to monitor the freezing process, but its main limitation is the shadow cone [60,61]. Its use can and often must be combined with the use of thermocouples placed in appropriate areas of the tissue.
The evolution by research  on the validity of computed tomography (CT) and Magnetic Resonance Imaging (MRI) to monitor the freezing of tissues, was made[62,63,64]. CT has the advantage of providing three-dimensional image. MRI in addition to providing a three-dimensional, with appropriate software is also able to predict the isothermal curves in the frozen tissue. The amount of destroyed volume was slightly lower than that frozen, as forecast, because the cells comprised in the range between zero to -20°C are not completely destroyed. Other less commonly used techniques have aroused interest in the various experiments including thermography and the impedance, including the recent technical development of electrical impedance tomography [65,66,67,68].

The cryogenic lesion.

Before the modern era in 1960, in studies of physiological processes freezing techniques have been used to destroy local tissues such as brain, heart, liver, kidney of rabbits and cats [69].
Scholars have described the nature of the lesion that is equipped with a central area of necrotic large size, surrounded by a narrow peripheral band or border partially damaged and with surviving cells. The peripheral area is a region of great interest because of the additional therapeutic implications. In this region, the cooling rate is slow, the duration of freezing is short, the final temperature is in the range between zero and -10°C and the heating is rather rapid. Some cells are in a state of necrosis, apoptosis and   others can survive. Those will go in apoptotic necrosis in anoxic conditions, typical of cryogenic injury [70,71,72,73,74]. Some cells survive even a  one thermal and osmotic stress of
-15°C [47,6,48]. It is in this range that it becomes important to study the different sensitivity thermo-physical of cells.

Sensitivity   to freezing   of the cells.

Different types of   cells have   a different sensitivity   to the   cooling temperatures .  These   can be identified   through the   short   cycles of freezing .  Experiments   show   the changes   of   sensitivity to cold   that occur   in the temperature range   between zero and   -30°C   [18 ,  75 ].  Were analyzed   sebaceous glands   and hair follicles   in   this temperature range ,  but   the keratinocytes   survived   at -30°C.   The   osteocytes   are killed   at about   -10°C [ 76].  In physiological conditions,   kidney cells , liver  and prostate   die   at about   -15   and   -20°C   [ 68,77,78,79,80].  Fibroblasts are   the most resistant   to freezing.   Even   cancer cells   show   an extraordinary and   variable   resistance   to the effects of   low temperatures [ 81,82,83,84].  The blood vessels , bone and  nerve structures   remain intact .

Wound healing.

The healing of the lesion is related to cryogenic impact severity and type of tissue. A lesion produced by a brief exposure to the cold of -10°C can lead to a loss of just tissue and heal quickly. At a temperature range of -20 to -30°C, the loss of tissue will be greater and directly related to the duration of exposure. Wound healing is usually complete. The process begins with an inflammatory reaction mediated by neutrophils and mononuclear cells, then stimulated by other factors such as prostaglandins, histamine and cytokines. In this cellular infiltration and edema follows hypothermia that develop with thawing of the frozen tissue. Scholars have suggested that the cellular infiltrate contributes to the development of apoptosis and tissue destruction [85,86]. The cellular infiltration helps to establish the new vascular system, which is fundamental to the process of tissue repair [76]. The slowness in healing is a characteristic of the lesion by freezing. Time is necessary to remove the necrotic tissue, both for "slough" (skin regeneration) or reabsorption that is definitely slower than surgical excision.

Different   tissues .

The different cellular tissues react in kind dissimilar to the stress of the cold. The skin is rich in collagen, elastin, and fibroblasts, which are resistant to the insult of the freezing, so the healing is generally favored. Damaged the collagen is slowly absorbed and replaced by the new one. The muscle fibers and the cellular tissues, such as the liver and the kidneys, can heal with the replacement of fibrous tissue, which can be a long process, according to the volume of frozen tissue. The architecture of the nerves, major blood vessels, and the bone is largely preserved after devitalization and serves as a scaffold for repair. Different tissue structures have been the focus of preliminary experiments for the clinical application of cryosurgery, commonly for the treatment of cancer.
The use of cryosurgery in some tissues is common in clinical practice and has little need for a new experimental work on animals.



Computerization of procedures.

Programming the cryosurgical treatment of a voluminous mass necessarily entails the use of probes the number of which should be proportional to the masses who want to destroy tissue. This circumstance complicates and limits, in general, the field of action of cryosurgery requiring the use of more bits cryogenic (as previously stated, arrived to use them eight and even more). Do not let us forget, however, that a high power cryogenic and the use of more probes inserted in the structure to be frozen  complicate further the adjustment of the system, this involves a careful knowledge of the equipment and its operation, but the fundamental fact implies perfect understanding of the thermal diffusion of the cold front.
The laws of diffusion of heat (in our case means  negative spread but nothing changes the physical and mathematical aspects of the phenomenon) have been properly studied and the simulation in the cryogenic propagation of biological tissues has been a great development thanks to the contribution of mathematical software applications.
Because healthy tissue and tumor cells have very similar thermal properties, the lowering of temperature should be targeted and the definition of the parameters that vary from operation and from patient to patient ( i.e., layout and depth of insertion of cryoprobes, temperatures they reached, etc...) must be carefully designed.

We make   our own the words   of Dr.   Giovanni   Giorgi   describing   our subject   with   clarity   and simplicity:

"However, if several cycles of freezing and thawing are lethal to portions of the tumor tissue, the healthy tissue in the same way it can be affected: for this reason, each cryosurgical experiment  must be thoroughly planned in order to minimize the consequences of the action of the cryoprobes  on healthy regions. Since healthy tissues and tumor cells have very similar thermal properties, the lowering of temperature should be targeted and the definition of the free parameters which vary from operation to operation and from patient to patient ( i.e., positioning and depth of insertion of the cryoprobes,  reached  temperatures etc.) must be carefully designed. When, through the use of some imaging modalities, will be reconstructed precisely the boundaries of the tumor region to be treated and have already been considered possible physiological indications coming from the medical staff, the problem of the definition of the free parameters of an cryosurgical operation becomes tied exclusively to the physical dynamics of the propagation of heat in the tissue. The determination of the most appropriate configuration parameters before each operation is called cryosurgery cryosurgical planning and is generally carried out by automated systems based on complex mathematical and computational tools.
From the mathematical point of view, the propagation of heat is, like many other physical phenomena, governed by differential equations. In general, the resolution of the differential equation of heat gives rise to a family of solutions: from these, can be extracted a (unique) solution to a particular problem by requiring the satisfaction of those which are called initial conditions and boundary conditions, relating to the specific problem.
To give an example, in the particular case of cryosurgical operation, the temperature distribution at a given instant will be provided by the resolution of a differential equation having, as an initial condition, the temperature at the initial instant of the whole tissue ( 37 degrees centigrade ) and, as a boundary condition, the temperature reached on the respective edges from each cryoprobe; each configuration of cryoprobes will give rise to certain boundary conditions, which will identify the temperature distribution relative to that particular configuration."

The introduction of special thermal needles placed in defense of healthy organs that should not suffer damage from the cold were simulated by allowing, through these equations, to construct mathematical models of very complicated phenomena. In these analytical models is considered the spread of heat in an environment in function also of the time. It is able, therefore, to know the time in the thermal distribution of the front cryogenic. The space is called the specific geometry of the equation that is properly entered into the model and must comply as closely as possible the information received from the medical x-rays.
To realize a logical-mathematical simulation as corresponding to reality is necessary, in addition to the geometry of the masses, to quantify the data concerning the temperature variations related to the  blood flow of tissue. For the implementation of all these parameters and process them into usable data in the planning and monitoring of cryosurgery it is necessary to use a software rather complicated [87].
It is in this environment that instruments with characteristics of good adaptability, supported by simulation processes in the pre-treatment and monitoring in the assessment of the immediate results, are subject to technological development.
We shall have occasion, in a special chapter devoted to the history of the development of physical-mathematical cryosurgery, to verify and ascertain how much progress has been made in this important area. We, however, know how long is the road to achieve perfection instrumental.

Physics, engineering   and   biology   applied to   cryosurgery.

The instruments used in cryosurgery, for their operation, in general, take advantage of the Joule-Thompson effect (as we have previously stated). This phenomenon, known at the beginning of last century, is characterized by the physical phenomenon that, when a gas is made to pass through a pipe to the end of which the duct narrows to become of a size such as to be considered capillaries (fractions of a millimeter) and, therefore, made to expand in this way, it causes a change in temperature. The change in physical state (sharp change in pressure and volume) you can assimilate (with good approximation) to an adiabatic change, draws heat from the outside because it was done "work" in the expansion, it follows a decrease of temperature in the immediate vicinity of the capillary.
Everything is influenced by a number of factors, among them plays a fundamental role the  physicochemical conditions of the gas used for the cryosurgical operations.
And to describe the design conditions that must be resolved by the manufacturers of tools for cryosurgery help us again the words of  Dr. Giovanni Giorgi they say about it:

"In general, the direct problem of determining the solution of a differential equation given the  initial and boundary conditions and the inverse problem of determining from the solutions of a differential  equation   corresponding conditions are radically different.
It can be shown that, in the case of the heat equation, the direct problem is always associated with a single solution that depends continuously from the data, which means that, in the case where there is a lack of precision in the boundary conditions, it will manifest content on the accuracy of the solution. The same statement is not, however, more true when it comes to the inverse problem for the heat equation:  here, in fact, small deviations in the temperature data can lead to huge errors in the definition of the parameters boundary and, moreover, cannot always be true that a solution to the inverse problem exists or is unique. These intrinsic characteristics of the inverse problem (i.e., absolutely independent of the mathematical tools) are what make it difficult to realize fast and effective tools that solve the problem of cryosurgical planning. In addition, when (as in cryosurgery) we have to do with biological tissues, the difficulty of the problem is heightened by the fact that the propagation of heat is regulated by non-standard equations that must take account of external contributions of blood and tissues surrounding the passage of state due to freezing ."

For some time the boundaries of the new frontier of the cold are plotted. On one side are the knowledge and respect of known laws of physics, on the other acquisitions on the biology of tumors and technological innovations. However, in this regard, as part of unexplored terrain, there is the computerization applied to research and monitoring of the biological effects during the use of biomedical equipment.
As we have previously stated the dimensional look of a tumor mass cannot be represented by a single mathematical equation. Against the thermal diffusion of the following configurations of spherical nature. In other words, the heat (and thus cold ) spreads according isotherms spherical whose center is occupied by cryoprobe. It is interesting to verify in analytical terms, the behavior of multiple heat sources arranged in various positions of the surrounding space and analyze the various curves at constant temperature that are to be formed. The mathematical structures used to represent these thermophysical geometries  at the end of the last century had not yet been prepared (or at least had not yet been made with these purposes).

An experimental and analytical approach can give interesting solutions. It became necessary therefore to research the right thecnical of which was to monitor a tumor mass prior to surgical operation and to determine, analytically, the best conditions for disposal and thermal intensity attributable to cryoprobes. This means evaluating the size of the mass to destroy in terms of geometric shape and quantity of cells. At this point the "software " of the system, according to the structure of the tumor, would determine the number and the relative position of cryoprobes [87].
The above comments regarding the possibility of a simulation cryosurgical operation at the end of the last century was still in gestation and only in the early years of the present century (2000) we have begun to catch a glimpse of the first studies to put forward as soon as possible.
It follows that, given the significant amount of parts in the field, the interdisciplinary approach is fundamental and indispensable for further advancing the knowledge of cryobiology, chemistry, physics and engineering associated with the cryosurgery.


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