SUMMARY. Temperature and time are two basic factors influencing the effect of heat on the human organism. The degree of resulting damage also depends on the anatomical organization of the skin and hypodermis. Sweat glands and the vascular supply, with blood flowing in corium and hypodermis, act as an effective thermo-regulators for the deeper structures.Under each fully developed necrosis there is a problematic transient area, also known as a zone of blood stasis. which corresponds to the partial damage caused by heat conducted into deeper structures. In this area during the first 3 days cells are selected according to the resistance to the thermal trauma.The basis genetic information of cells is very resistant, but disorders develop on the genetic expression level. Cells - mainly fibroblasts - which survive the first selection are damaged by the thermal injury to varying degrees and often cause other complications. During synthesis of transcripts of RNA from DNA chains an excessive amount of transcripts can develop, subjecting the receptor to information about the loss of skin firmness in defective feedback to the CNS, blocked by fixed trauma emotion. The status is accompanied by swelling, lymphatic stasis, capillary stasis, changes of the local pH and others. During repair facilitated by inflammatory process, excessive amount of collagen is created, as has repeatedly been proved in experiments. The problem can be partially solved by early compression, which limits the amount of impulses about insufficient firmness of the skin, and improves the circulation. while reducing edema, normalizes pH and optimizes production of transcripts.RNA polymerase lacks the ability to correct perfectly and in fact frequently makes mistakes, even under completely normal physiological conditions. If the pH is wrong, it can make even more mistakes and produce pathological collagen in excessive amounts.RNA is not intended to preserve information permanently, and after a certain time it degrades. The onset of this degradation is determined by the cell as well as the amount of created proteins. If RNA is not degraded on time, overproduction of protein and collagen is a natural consequence of the developed defect.Messenger RNA (mRNA) directs the creation of proteins. In case that it is not properly cut in the cellular nucleus, qualitative and quantitative errors in transcription into the protein develop.The non-information RNA takes an enzyme part and plays a role during the transfer of RNA into the protein. It does not have the correction ability.Transfer tRNA chooses appropriately amino acids and places them into the growing protein chain. At the same time, it can make errors and interchange amino acids.
Key words: burns, physiology of burned skin, pathology of skin, genetic expression, scarring process, collagen, keloids and hypertrophic scars
Temperature and time are two basic factors influencing the effect of heat on the human organism. The extent of the resulting damage also depends on the anatomical organization of the skin and hypodermis. Sweat glands and the vascular supply, with blood flowing in corium and hypodermis, act as an effective thermo-regulator for the deeper structures. On a long-term basis, the human organism can survive extreme temperatures, either high or low. The ability to survive depends on the degree of training of the adaptive mechanisms. and on the psychological tolerance of the individual. After exposition the excessive heat is radiated out of the organism into the external environment, exhaled front the lungs or evaporates with sweat, or alternatively with water during active cooling of the skin surface.
When we consider the conditions necessary for tile development of skin burn, we have to mention special situations where-under precisely established conditions -a person can remain for a relatively long time without burn injury. One of these is the Finnish sauna, where a person can stay for as long as several minutes without skin burns.
The word sauna is Finnish. describing a small house or room used for this purpose after being heated by a wood or electric stove. The room warms up to a temperature of between 60 to 120 °C but there are also saunas working at temperatures 130 or 140 °C. In the Finnish sauna stones arc placed on the stove and then water is poured on them to temporarily increase the humidity of the air. The relative humidity in the sauna is low: usually several tens of percent (16).
This is a paradoxical situation. because water at a temperature of 80 °C causes burns of second to third degrees: however, in the much warmer environment of the sauna the skin is not damaged.
To explain this, we have to use our knowledge of anatomy, function of the skin and hypodermic as well as physical conditions of the environment in sauna. The air in the sauna is very dry, even though water is poured on to the stove. At the same time, the air is virtually still, allowing enough time for the creation of a cooler microclimate above the surface of the epidermis. Intensive sweating and water evaporation leads to fast cooling of the skin and cooling of the thin layer of the air around the skin surface. Capillary vasodilatation in the stratum papillare contributes to further cooling, while increased blood circulation cools deeper skin structures and increases water supply to the sweat glands. If the organism were exposed to agitated hot air, a burn injury would certainly develop.
Similar temperature conditions to the Finnish sauna exist at several places around the world. Human beings can survive even there; for example, in Death Valley, Nevada, USA.
The valley is 225 km long and up to 26 km wide. In the middle the bottom of the valley is below sea level, and there is almost permanently absence of wind. Here the measured temperatures reach extreme values; the highest measured air temperature in the shade is 57 °C, and usually it is above 50 °C. Despite these facts, it is possible with enough drinking water to survive here on a longterm basis. Similar conditions exist in equatorial desert areas which are permanently inhabited by people (4).
The other extreme is represented by the cold environment of areas close to the Earth poles, also permanently inhabited by people. Temperatures exceed -40 °C in winter and can fall to as low as -80 "C in extreme periods. Again, the thin protective layer of air above the dermis maintains an acceptable temperature and helps to prevent freezing of the cells. Clothing or fur in animals keep the cold weather microclimate close to the surface of the skin and prevent excessive temperature losses and hypothermia. However, it the freezing air is circulating, even very experienced polar explorers can develop frostbite or die due to hypothermia.
During heat transfer via conduction in metals and homogenous objects, simple physical values can explain the phenomena, such as the square of the distance from heat source with which temperature decreases in a substance. In living tissue, particularly in the skin. (his rule does not apply exactly, due to the sweat glands and vascular plexuses in dennis, where the square distance is significantly limited in reaching into the deeper structures. However, it is only limited, not completely eliminated. A major and very significant role is played by the time factor. If temperature increases gradually, circulation reacts with vasoditatation and efficiently cools down the epidermis, alongside increased sweat production and sweat evaporation. However. if the temperature increases quickly (boiling water, steam, fame, electric arc), the blood vessels and sweat glands cannot react, and the cooling effect in the first seconds is accomplished only by evaporation of water from epidermal cells. When the water evaporates, the cells dry up and char - and, depending on the time of exposure to the high temperature, deeper structures are similarly impaired. If the exposure to high temperature is long, dermal plexuses are impaired and react by vasoconstriction of arterioles and microthrombosis of venulae. The effect of cooling by blood circulation is eliminated: sweat glands are unable to react and necrotize. A full skin burn develops. Under each fully developed necrosis there is also a socalled transient area, or a zone of blood stasis, which corresponds to the partial damage caused by heat conducted into deeper structures. In this area. over the first 3 days, cells are selected according to their resistance to the thermal trauma. This process is completed 3 days after the injury. The earliest we can describe the extent of damaged tissues in clinical practice is only the third day after the trauma. In extensive damage even this interval is not final and development of burns continues further according to the course of the post-trauma shock, possible infection complications, etc. The health condition of the patient prior to the trauma, psychological status of the burned patient and other factors apply. Again. this confirms the point that time plays a key role on the development of a burn.
However, cells - mainly fibroblasts - which survive the first selection arc damaged by the thermal injury to varying degree and often cause other complications, which we will mention in more detail.
Cell biology teaches us that genetic information in cells is transferred from DNA through RNA into proteins, a process called gene expression. After the selection of genetic information saved in the DNA, the nucleotide sequence is transferred into the RNA, and this transcription is catalyzed by the RNA polymerase. The nucleotide sequence contains information about where the transcription should start and finish. RNA differs from DNA in the content of the glucose part (ribose instead of deoxyribose) and base (Uracil instead of Thymine). In the cellular nucleus it is synthesized as it shorter onefiber molecule which often packs into a three-dimensional structure.
In cells several types of RNA develop: information (mediator. messenger) mRNA, which contains information for synthesis of protein; ribosomal rRNA which is part of plasmatic ribosome; and tRNA, which is an adaptor during its own proteosynthesis.
In eucaryotic cells the majority of genes in DNA are formed by a large number of short coding sections, socalled exons, divided by longer non-coding sections introns. During the transcription both sections are transcribed into the primary mRNA. Introns are removed from the primary transcripts in the cellular nucleus by a process called RNA splicing and oxen sections are interconnected. This way the spliced rnRNA is transferred into the cytoplasm.
In the cytoplasm there are big ribonucleoprotein elements called ribosomes, and here the mRNA is translated into the desired protein. One amino acid, given by the genetic code, is attributed to each of the codons, and therefore by a combination of four various nucleotides we can get 64 different codons. The majority of amino acids are determined by more than one codon.
During enzyme proteosyntbesis itself the tRNA acts as adaptor. Complete protein is released only after the translation stops on one of the end codons.
Degradation of proteins in cell is carefully regulated, and some proteins are degraded in cytoplasma by the great protein complex - proteasome. Even though the flow of information in cells is DNA-RNA-protein, some very important reactions are permanently catalyzed by developmentally older RNA (1).
For the sake of simplification, in the list of individual degrees complicated names of enzymes are not listed which complete placement of relevant bases and amino acids to proper positions.
This basic cellular genetic information is unfortunately indispensable, because if we want to understand at least partially the process of healing and particularly scarring, we find many works pointing out that the scarring failure is based on genetics; firstly. there is open space for faulty transcripts from DNA to RNA. It has been found that physiologically RNA transcript is faulty by 1(N (!). because the particular transcript enzyme - RNA polymerase - does not have a reparative mechanism. (DNA has polymnase reparative mechanism, because the spontaneous change of basic genetic code is inadequate and therefore errors in nucleotide transcript arc very rare, only 10-7(!)).
Elementary cellular function is under control of the genotype itself. The genotype reacts by expression of individual genes to the signals from the environment, coming mainly from surrounding cells through intercellular environment, and we have to consider central nervous and endocrine function regulation of all organs as well as cells. With the name genetic features, the behavior of epithelial, muscular, ligamentous and parenchymatons organ cells or neurons differ. Cellular communication is completed through signal substances, which if soluble in water do not penetrate through the cellular membrane, but activate receptor proteins on the surface of cells and collocate with them. These receptors then transmit signals through the plasma membrane into the cells. where they are transported by various systems into the nucleus. There they activate or suppress gene expression. Through the membrane only hydrophobic molecules of steroid hormones or nitrous oxide can penetrate and activate the receptor proteins inside cells. There are three types of cell surface receptors with various reactions to the impulse; however, more details would exceed the scope of this publication (I).
Four basic types of intercellular communication have been documented: humoral, completed primarily by hormones affecting the whole body; these arc produced into blood circulation by endocrine glands, next by local cellular rnediatars, influencing a small group of cells in the immediate surroundings-used mainly in local inflammation and during healing of wounds and defects. Other communication activities between immediate neighbor celtc and mediators are transmitted directly through cellular membranes. This type of communication exists for example between kcratinocytes in stratum spinostrm of the epidermis; however; it mainly dominates in the embryonic time, when cells inform each other mutually about their position in the embryonic body and neighbor cell reacts to the impulses according to the established genetic program to prevent parallel formation of the same organ or duplication of its parts. Specific interaction between cells is formed by transmission of information through neuron fibers from distant neurons of central nervous system. The advantage of this transmission of information is in the speed of conduction in the neuron fiber (up to 100 m/s). Certain disadvantage after burns can be impulses inadequate to the local situation when accompanied with a strong emotional component during the injury.
The basis genetic information of cells is very resistant. The double spiral of the cellular genome can withstand temperature up to 90 °C, and hydrogen bridges divide only in values of pH 9-10. Therefore, compared to other cellular structures it is very resistant against damage. This is the fundamental of a cell, the principle of life, and it has to be safely transferred into next generations. Other mechanisms ensuring the cellular function are not so resistant.
Examples of failures that can develop after a burn trauma
During synthesis of transcripts of RNA from DNA chains, excessive amount of transcripts can develop, subject to the receptor information about the loss of skin firtnness in defective feedback to the CNS, blocked by fixed trauma emotion. The status is accompanied by swelling. lymphatic stasis, capillary stasis, changes of the local pH and others. During the reparation inflammatory process an excessive amount of collagen is createdthis has repeatedly been proven experimentally. The problem can be partially solved by early compression, which limits the amount of impulses about insufficient firmness of the skin, improves the circulation while reducing edema. normalizes pH and optimizes production of transcripts.
RNA polymerase does not have the perfect ability to correct and frequently makes mistakes, frequently even under completely normal physiological conditions. If the pH is wrong, it can make mistakes even more frequently and produce pathological collagen in excessive amounts. RNA is not designed to preserve information permanently, and after a certain time it degrades. The time of degradation is determined by the cell as well as the amount of created proteins. If RNA is not degraded on time, overproduction of protein and collagen is a natural consequence of the developed defect.
Messenger RNA (mRNA) directs the creation of proteins. In case that it is not properly cut in the cellular nucleus, qualitative and quantitative errors in transcription into the protein develop.
The non-information RNA takes an enzyme part and plays a role during the transfer of RNA into the protein. It does not have correction ability.
Transfer tRNA chooses appropriately amino acids and places them into the growing protein chain. At the same time, it can make errors and interchange amino acids.
Collagen is an extracellular scleroprotein not soluble in water. It contains large amounts of glycine, proline and non-coded amino acid hydroxyproline, which stems from postrranslational modification - enzyme hydroxylation. Collagen, like other proteins, is formed by about 20 types of basic amino acids. A characteristic typical for collagen is its firmness, provided by the triple chain spiral structure of molecules, reinforced by transverse bands. It is a structure protein with characteristic arrangement of amino acids - on every third position of its macromolecule there is amino acid glycine. allowing for its specific structure in space. In human genome more than 40 possible collagen genes have been found. and about the same amount of genes that code shorter or longer sequences of collagen, similar polypeptide chains. Collagen generally creates white, non-transparent fiber formations that are wrapped by various amounts of proteoglycanes and other proteins (according to the type of tissue). Collagen fibers are microscopically easily identifiable due to the cross-banding, possibility to dye histologically, possibility to swell and a sudden contraction when under 60 °C.
The most common is Collagen Type h present in skin, bones, ligaments. ere. Its amino acid composition, as well as the detailed sequence of amino acids in its two different polypeptide chains, is already known.
Type II is present in hyaline cartilage and intervertebral discs, and its main characteristic is relatively high content of hydroxylysine - 23 remainders to 1000 amino acids -compared to the type I, where are 5 remainders to 1000 amino acids.
Tpe III occurs with type I in skin as reticular fibrils and also in smooth muscles. From a chemical point of view, the typical aspect of type III is a higher content of hydroxyproline (124 remainders to 1000 amino acid remainders) and also the presence of cysteine, which stabilizes collagen structure by covalent disulfide linkage (cystine).
Type IV is present in basal laminas, and like type III it has a higher content of hydroxyproline (135 remainders to 1000 remainder of amino acids); it also contains cysteine and has a high content of hydroxylysine.
Type V can be found in basal laminas as well as in dermis, tendons, bones and fibro-cartilage. In embryonic tissues and during some healing processes a non-normal type of collagen that develops as a formation of three polypeptide chains type I has also been identified (7). Collagen fibrils and organic components of matrix are produced and formed by fibroblasts. Fibroblasts partially directly organize fibrils, orient them and - according to need-rebuild them. Collagen molecules are secreted in the form of protocotlagen with additional pcpfides on both ends, preventing their organization into fibrils. Extracellular enzyme collagenase eliminates the end peptides only after they tire securely outside the cell and the so-called tropocollagen polymerases into collagen fibrils. At various body parts the fibrils have various structures of organization to ensure optimal firmness in the desired directions. This is secured by fibroblasts, which move on collagen fibrils, stretch them and strengthen them into flat leafs and ligaments. Polysaccharide and protein gels fill out the free space and ensure compressive strength. These are primarily negatively charged and various molecules of Oycosarninoglycanes (GAG), mutually connected into huge macromolecules. Its content varies according to the type of tissue. In tendons there are primarily collagen fibrils. in the vitreous burnout there are virtually no collagen fibrils. Molecules GAG are strongly hydrophilic and occupy a huge space. They also contain many negatively charged particles, which attract osmotically active Na•, and therefore great amount of water is sucked into the matrix. The developed swell pressure is in balance with collagen fibrils.
Proteoglycanes also have another function. They filter passage through matrix, fixate growth hormones and other proteins that serve as signals for fibroblasts and take control of cell migration through matrix. That is how cells of matrix mutually influence themselves and establish cellular differentiation.
Skin collagen is formed mainly by two types of collagen, type I and type IIT. Mammals have about 20 genes available for collagen coding and code its various forms according to the need. In mammals 25% of proteins in the body are formed by collagen. Collagen is formed by three chains of linear molecules twisted into a spiral like a rope. Particular macromolecules of collagen are formed by fibrils with diameter of 10-300 not and many micrometers long. Fibrils form filaments. Particular fibrils are mutually interconnected and connected with extracellular matrix by collagen (1).
Mammals have more than 200 various types of cells, the majority of which are epithelial, where cellular membranes are tightly packed and often form layers of multiple cells. Skin epithelium is layered or multilayered. Cells are primarily flat, like files.
For a multiple cells organism these have the same importance as the plasmatic membrane for a cell. Epithelia are polarized and lay on basal lamina. Basal lamina is a thin layer of extracellular matrix, composite of collagen type 1V and various other molecules (laminin and others). Apical and basal side of epithelial cells always differ in structure and function. Tight connections in walls ensure epithelial insolubility. Other connections are mechanical (adhesive), interconnected with cytoskeleton (desmosomes); the cell is fixed to the basal membrane by hem ides mosomes. These fixation proteins are called cadberins and require presence of Caz* ions in extracellular environment. The intracellular net, composed of actin and cytokeratine filaments, is then interconnected throughout the whole epithelium. The net can contract and the epithelium can produce pressure or change its shape. This function is very important. especially in the embryonic stage when many epithelial exaginations form specialized organs, for example the neural tube. crystalline lens pouch and others. Cytoskeleton of dermal epithelium is formed by keratin, whose filaments cross the cytoplasm and are mechanically very resistant. Cells are fixed to the basal membrane by integrins, proteins of basal plasmatic membrane of epithelial cells. Inside the cell they are again fixed to the keratin skeleton and are called hemidesmosomes. Interspaced connections between cellular walls called nexes allow exchange of ions and small molecules between the cells. They contain protein complexes that are often called connexons and overlap cellular interstices by narrow ducts that allow for passage of ions and small molecules soluble in water up to size of approximately 1000 daltons. That is how cells are electrically and metabolically interconnected (1).
From the anatomy of skin we know that the surface of skin is supplied not only by sebaceous glands but also by glycolipids produced in the stratum granulosum of the epithelium. Furthermore, the surface is moistened by sweat, and the precipited salts keep the corneal surface layer osmotically continually active, moistening the environment by absorption of air humidity. That is why the corneal layer of skin is permanently soft, flexible and resistant against mechanical damage.
The dermis is not fully functional in scars that have spontaneously epithelized after deep burns. At the attempt to rehabilitate numerous bulls form, because the papillary layer is initially not completely formed and it is partially restored during the first months of scar maturing. The scar then ensures firm connection between epithelium and corium and the bulla and abrasions cease to form. Stratum granulosum is restored relatively late; the adnexa almost does not regenerate, sometimes these are at least transferred by an autotransplant and with it is also transplanted a layer of fully functional skin with differentiated epithelium and elastic fibers in corium. That is why transplanted surfaces mature 100% faster than areas left to spontaneous epithelization and arc cosmetically much more optimal (only if the autotransplant is not widely meshed). In spontaneously epithelized scars of deep areas, collagen fibers form almost without the elastic fibers, which after scarring manifests the stabilization by the wrinkled surface of the scar, like in older people.
What influences course of healing
Keloids and hypertrophic scars
Two hundred years ago, J. L. M. Alibert (8) first pointed out that there are differences between keloids (K) and hypertrophic scars (HJ), and arguments about their classification continue until today. J. L. M. Alibert (8) claimed that K is more painful. the pain is sharp, stabbing, burning and they often develop spontaneously. HJ develop after inflammation, burns or ulcers. These criteria are still basically accepted today. In the 19th century HJ became an independent clinical unit. when Kaposi in 1874 postulated that scars formed at the edges of skin loss injury are hypertrophic even though they look like keloids. However, he did not know the cause, and this principle was only philosophically disputed. A great deal of work on microscopic analyses was performed, and even electronic microscopes were used, until the conclusion was reached that from a morphological point of v icw there are not many fundamental differences between the two. In 1996, Linares (8) proposes to set up a team of specialists, histochemists, histomorphologists, imntunohistochemists, biochemists, immunologists, and a laboratory with tissue cultures, in order to systematically research cells, matrix and mutual interactions. In 1996 he did not anticipate that the fundamentals of the problem lay deeper, and that without genetic analyses this question cannot be successfully solved (pp. 347-379).
We know that for a burn to leave a persistent effect the damage has to exceed a certain borderline, and that is the papillary stratum of the corium. Under the necrosis there is a very problematic area, zone of blood stasis in capillary blood flow. where cells - primarily fibroblasts - arc impaired partially. From the necrosis towards the deeper healthy tissue there is less and less damage, and if this layer is left untreated it becomes the source of later problems. According to recent studies. published in renowned journals and publications, it seems that the proved cause of hypertrophic scars is excessively long survival of high number of fibroblasts due to the decreased number of apoptotic proteins, responsible for optimally programtned cellular death and at the same time corresponding by fibroproduction of the collagen fibers. Apoptotic proteins in fibroblasts are exprimated by normally present genes; so far 64 of the genes have been detected. In 8 of the main genes it was found that their activity decreased by 18-51 % of north (11). Next, it is imperative to mention dysfunction of gene expression in the poorly working fibroblasts that were selected from keloid and hypertrophic scars and compared with fibroblasts gained in a normal dermis. It was found that there is a dysfunction on two levels of gene expression and that there are two mechanisms regulating the collagen type 1 synthesis, one transcript and the other posuranscript. In keloids as well as hypertrophic scars the gene expression is increased by transcription from DNA x1 (l) of procol- with subsequent increase of coding mRNA for x1(1) of protocollagen, which forms cotlagen 1. The difference between both types of scars is that to kelnids tile described status is permanent In contrast, in hypertrophic scars after a certain tune the process is leveled on the mRNA level for x1 (l) (1) procollagen or a posttranscript level, and the process is stabilized. Furthermore. in keloids with overproduction of collagen 1, production of collagen Ill also increases (5).
Another very interesting finding known front clinical experience and lately confirmed in laboratory (14) is the positive influence of epithelial inhibitors on fibroblast proliferation. Reality speaks for timely excision and an immediate re-cpithelization of the area by a thin dermoepidermal graft with very good postoperative results. A very important role is played by cytokines and mainly factor TGF-f3 (transforming growth factor beta), which supports growth and proliferation of fibroblasts (12, 15). In some cases, even laboratory and cytology evaluations talk about allergic effects (10, 17).
Myofibroblasts that develop by transformation of fibroplasts are frequently studied. Myofibroblasts have contractile abilities and the ability to fixate, and subsequently they pull the newly formed collagen nets and contribute on the development of scar contractures (13). Even here authors have attempted to influence the phenotype transformation of fibroblasts to myofibroblasts by the growth factor FGF-2 already in the phase of granulation and succeeded.
Another interesting area of cellular research involves comparison studies of fibroblasts gained from areas of body, where the hypertrophic scars do not form (buccal mucosa) and comparing them to the other body areas. According to Okazaki (9), fibroblasts from buccal mucosa are equipped by a higher expression of growth factors, and defects heal faster than in other parts of body.
Very much research has also been performed on the role of free radicals and lately mainly nitric oxide (NO): the resulting activity can play a key role in the development of hypertrophic and keloid scars (3).
According to Friedman (5), who researched the gene expression of collagen 1 and III in fibroblasts from keloids, hypertrophic scars and normal skin, the fibrin overproduction problem comes from failure oil two levels of transcript of collagen 1 and 111 gene formation. There are significant differences between fibroblasts from keloid, hypertrophic scars and normal skin. Keloids have significantly increased rate between collagen type I and type III accompanied by permanently increased gene expression and increased rate of transcription x1 (I) of procollagen gene. In hypertrophic scars the situation is similar, but posttranscription mechanism can decrease high levels of mRNA which code the x 1 (1) procollagen. In normally functional fibroblasts this situation does not occur. However, the causes of such disorders have not yet been clarified, and we have to speculate based on clinical observations.
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