Cooling and Hypothermia Effect on Structural and Metabolic Characteristics of Bone (Review)

The review presents the basic concept of hypothermia and its classifications. The findings of hypothermia effect on cultured cells (osteoblasts and osteoclasts) as well as the effect of cold stress and different regimens of hypothermia on structural and metabolic characteristics of bone were analyzed.

According to the principles there are primary and secondary hypothermia [31].Primary hypothermia develops in healthy people under the influence of environmental factors, aging, and pharmacological agents, secondary one occurs as a result of disease complications [32,36].
In clinical study performed by M. Mallet [32] in the patients with mild hypothermia it has been emphasized that 85% of them were older than 60 years.With age, the body temperature reduces because of different reasons: an organism's ability to produce heat [3], decrease of thermogenesis activity in brown adipose tissue, peripheral vascular constriction [13,38], inhibition of metabolic processes in an organism [37].In older people the remodeling processes of bone tissue are inhibited with a predominance of resorption over osteogenesis, accompanied by the development of osteopenic and osteoporotic disorders [27,50].Similar regularities were found in aged animals [41,42].
Aging is accompanied with an inhibition of metabolism and energy exchange, resultung in reduction of adaptive capabilities of an organism.This is primarily due to the changes in protein synthesis.There are disorders in metabolism of carbohydrates and lipids, associated with an activity change of certain enzymes and oxidative processes as well as the accumulation of lipid peroxides in tissues.
Metabolism is inhibited due to hypothermia, which can lead to the development of hypothermia [14,32].It has been established that reduction of body temperature by 1°C is accompanied by 6% slowdown of general metabolism (it is determined by reduced indices of oxygen consumption) [55], due to the decrease of protein, carbohydrate and lipid metabolism.Under hypothermia it has been noted the weakening of fasting metabolism [14,32].There is disruption of respiratory function of blood, oxidative processes and acid-base balance.The combination of these factors (on a background of chronic hypothermia) leads to the changes in regulatory mechanisms and can cause pathological changes in структурно-метаболічних порушень як у клітинах суглобового хряща (пікноз ядер хондроцитів, лізис окремих ділянок цитоплазми, редукція ендоплазматичної сітки, апарату Гольджі, руйнація крист мітохондрій), так й у міжклітинному матриксі (зміна складу протеогліканових комплексів, набряк та демаскування колагенових волокон) [8,37].
Hypothermia effect on bone tissues has been currently examined in two ways: at the level of the body, and in cell culture (osteoblasts and osteoclasts) enabling to assess structural and metabolic peculiarities of investigated object without affecting endogenous factors of an organism.

Effect of hypothermia on cultured cells
In cell culture it is possible to simulate the effect of temperature parameters, which satisfy hypothermia conditions observed in humans and animals.
The effect of mild (35.5°C) and moderate (34°C) hypothermia was investigated for 14-16 days in cultured osteoblasts isolated from calvaria of newborn rats, and osteoclasts derived from the culture of osteoclast-forming monocytes/macrophages of bone marrow of 6-8 weeks old mice [38].It was found an inhibition of proliferation and differentiation of osteoblasts under given regimens of hypothermia.The number of osteoblasts in culture after 14 days of culturing in moderate hypothermia (34°C) decreased by 30%.In addition, biosynthesis of alka-line phosphatase, osteocalcin and collagen of type-I decreased.Bone nodule formation by osteoblasts cultured at 34°C during 16 days decreased by 95% and at 35.5°C reduced by 75% if compared with the control culture (37°C).It is known that under hypothermia (32°C) in fibroblasts culture a mitotic cycle is disrupted by lengthening G1 phase [37].J. Patel et al. [39] suggest that another mechanism of inhibition of osteoblasts' proliferative activity may be the dysfunction of TRPV receptors (Transient Receptor Potential Vanilloid) channels, whereas osteoblasts, in particular, express different receptors of these channels (TRPV2 and TRPV4).It is possible that TRPV channels are temperature sensors of cells [1], but this hypothesis requires further study.
На відміну від вищенаведених даних щодо активізації в умовах гіпотермії остеокластогенезу of mild hypothermia the osteoblasts remain viable and biosynthetically active, however, the fluorescence intensity of actin fibers of cytoskeleton, involved in motor activity of cells and cell organelles increases.After exposure of severe hypothermia to cultured osteoblasts there were observed more expressed changes (if compared with the effect of moderate hypothermia) in the structure of cytoskeleton.Actin fibers with an intensive fluorescence are localized around the nucleus, but normally they are placed homogeneously in the cytoplasm and exhibit slight fluorescence.There were no changes in tubulin structures (microtubules).Moreover there was ob-served a decrease in alkaline phosphatase and os-teocalcin activity as well as expression of CD44 (surface cell glycoprotein playing an important role in adhesion and cell migration).It was revealed that increasing the term of mild hypothermia effect up to 24 hours resulted in the appearance of apoptotic cells, which number was by 3.54% higher if compared with the control culture, herewith the osteoblasts remained metabolically active [3].Under severe hypothermia the number of apoptotic cells increased by 13.2%, there was also observed a decrease by 56.7% of biosynthesis with osteoblast proteins.
Extending the culturing of osteoblasts up to 5 days under severe hypothermia (27°C) resulted in a significant reduction in mRNA, which encodes the matrix protein SPARC (secreted protein acidic and rich in cysteine).It plays an important role in formation of extracellular matrix, as well as in regulation of proliferation, adhesion and movement activity of osteoblasts, that is a significant part of osteogenesis.With its participation balancing the processes of bone formation and resorption occurs [2].During hypothermia there was observed a reduction of SPARC expression and cell proliferation.
When culturing for 16 days mononuclear precursors of osteoclasts on ivory discs with the addition of the factors, stimulating osteoclastogenesis such as M-CSF (macrophage colony-stimulating factor) and RANKL (receptor activator of nuclear factor kappa-B ligand), it was established that the number of osteoclasts significantly increased in the cultures after exposure of mild (35.5°C) and, in particular, moderate (34°C) hypothermia if compared with the control ones (37°C) [38].There was also increased the surface of resorption discs on which mononuclear precursors of osteoclasts were cultured, testifying to differentiation and activation of osteoclasts.The number of osteoclasts and resorption lacunae increased in 1.5 times (at 35.5°C) and 2 times (at 34°C) if compared with the control culture.The authors believe that the results obtained in cell culture suggest a potential negative impact of hypo-S.Meghji та співавт.встановили, що за умов культивування остеобластів (клітинна лінія остеобластів MG63) упродовж доби (за температури 33°С) відмічено підвищення біосинтезу остеопротегірину, в той час як експресія рецепторів RANKL залишалася на тому ж рівні, тобто створювалися умови для пригнічення активізації остеобластів [45].
Contrary to above mentioned data as for activation of osteoclastogenesis under hypothermia S. Meghji et al. established that during culturing of osteoblasts (MG63 osteoblasts cell line) for a day at 33°C there was observed the increase of osteoprotegrin biosynthesis, while the expression of RANKL receptors remained at the same level i.e. the conditions for inhibiting the activation of osteoblasts were created [34].
Thus the results of research carried out in cell culture indicate hypothermic effect of various intensity on bone cells that are involved in its remodeling (osteoblasts and osteoclasts).Let us consider how hypothermia affects bone cells (proliferative activity, ultrastructural organization, metabolism peculiarities) in organism, on the structure of bone tissue matrix as well.

Effect of cooling and hypothermia on bone tissue
Nowadays the mechanism of hypothermia and low temperatures effects on bone tissue have not been completely understood.There are the data that during influence on organism of different types of stress, including cold, the activation of free radical oxidation of lipids [44,49] occurs, resulting in disorders of hormonal regulation, increasing flexibility of cell membranes, reduction of their mobility, destruction of lysosomal membranes [23].This, in turn, may be the cause of destructive changes in bone tissue [24,54].The bone tissue state was investigated under conditions of cold stress (without determination of body temperature) and hypothermia of various regimens (with determination of body temperature).
Доведено, що тривалий холодовий вплив значно збільшує концентрацію мікроелементів у кістковій тканині дослідних тварин порівняно з інтактною membranes, reduction of density of ribosomes), mitochondria and nucleus structure.A number of lysosomes in cytoplasm was significantly increased.In mitochondria there were observed expressed destructive changes such as swelling and lysis of mitochondrial membranes and cristae in some areas.These disorders affect the energy supply of cells that is one of the mechanisms of their destruction and death.It is proved that hypothermia negatively affects the state of mesenchymal stromal bone marrow cells, inhibiting their proliferative activity during culturing and ability to form cell colonies.
In a compact and spongy bone of both young and aged rats strong disruptive disorders such as decrease of osteocytes density on the surface of bone trabeculae, osteocytes lacunae expand, osteolysis, fibering of bone matrix with collagen fibers retrieval, cracks and crevices were revealed.Destructive changes were significantly higher in bone tissue of old rats.Analysis of morphometric indices of bone tissue remodeling showed that hypothermia of the given regimen activated osteoclastogenesis and inhibited osteoblastogenesis, leading to the prevalence (especially in aged rats) of bone resorption [41,42].
There was studied the effect of prolonged cold effect (the rats were maintained for 14 and 30 days in climate-chamber at -15°C for 3 hours a day) on microelement (Mg, Ca, Al, P) composition of thigh matrix, while the body temperature of animals was not controlled [24].Under cold stress there was revealed a disturbance of mineral metabolism in bone: to the 14 th day Mg content increased by 37.6%, and after 30 days it decreased by 4.8% from the initial one, Ca level to the 14 th day increased by 13.7%, and after 30 days it decreased by 12.6% if compared with the control indices.The concentration of Al in this period was by 3.2% higher if compared with the control indices, and after 30 days the one decreased by 32.3%.The level of P in bone to the 14 th and 30 th day was significantly reduced (by 67.0 and 68.1%, respectively).There was revealed a change in the content of lipid peroxidation products.Significant accumulation of diene conjugates (in 1.68 times higher if compared with the control) was observed 14 days later cold exposure on rats.
In experimental study of A. Riesenfeld [43] the reduction of cortical layer thickness of rat long bones to the 7 th day after cold effect (swimming animals in cold water (8°C) for 10 min during 10 days) was established.Inhibition of growth processes in epiphysial cartilage due to chondrocytes death and destruction in epiphysis of long bones of rabbits under cold stress (cooling limbs of young rabbits in ethanol (96°C) down to 20 and 15°C) resulted in a significant reduction of limb length [11].
B. Steinberg et al. [48] studied the effect of cold stress (staying in a cold chamber at 5°C) for 7, 15, 21, 50 and 90 days on a state of hamsters' thigh.Control animals were kept at 27°C.Histomorphometrical analysis showed that after 90 days of cold stress there were changes in structural organization of bone tissue: increased number of resorption cavities filled with osteoblasts; expansion of lumen of the central channels of osteones; decrease of osteones number and surface of bone trabeculae.The observed changes in bone tissue were referred by the authors to osteoporotic disorders.
Metabolic changes in bone tissue were studied under cold stress.In the experiment performed by P. Patterson-Buckendahl et al. [39] the rats were cooled (cold chamber -8°C) for 3 weeks (1.5 hours per day), and then there was revealed a decrease of osteocalcin by (21.8 ± 1.4)% in plasma if compared with the control, indicating the inhibition of biosynthetic activity of osteoblasts and deceleration of remodeling bone matrix whereas the synthesized osteocalcin by osteoblasts was accumulated in the organic component of bone matrix.
Along with the studies performed in the animals without pathology there are the reports, wherein the hypothermia effect under conditions of metabolic bone disease, namely, osteoporosis has been evaluated.In the animals with experimental osteoporosis (resulted from ovariectomy as a model of postmenopausal disorders in women) there was studied the effect of cold (forced swimming of rats in cold water (8°C) for 5 min during 7 days) on calcium metabolism and its removal of a bone [21].It has been established that in the rats with osteoporosis exposed to cold stress the content of corticosteroids, thyroxine and thyroid-stimulating hormone in blood plasma significantly increased if compared with both control (intact) rats and the ones with ovariectomy, not exposed to cold stress.There was revealed a reduction (in average by 31.8% compared to the rats with ovariectomy without cold stress) of Ca 2+ transport through intestinal mucosa and a statistically significant decrease of alkaline phosphatase activity and calcium dependent ATPase (Ca 2+ -ATP-ase).In blood plasma of experimental rats (after cold effect) the indices of alkaline phosphatase and concentration of Ca 2+ were significantly higher if compared with the control rats and the ones with ovariectomy without cold stress.The authors conclude about the negative impact of cold stress on bone tissue under conditions of estrogen deficiency [21].
It is known that calcium increase in blood is accompanied by its accumulation in cells of a body.According to the hypothesis of P. Hochachka [19] under hypothermia the factor destabilizing the cell metabolism and leading to impairment of its function is ionized Ca 2+ , accumulating in cytosol.Due to the opening of voltage-dependent Ca 2+ channels and rapid accumulation of Ca 2+ the phospholipid hydrolysis of membranes and irreversible cell dama-ge occur [12,47].The given mechanism of cell destruction may be characteristic for bone cells, but this hypothesis requires further research.
Under hypothermia caused by cold effect, not only morphological and metabolic abnormalities in cells and matrix of bone tissue, but also a series of negative changes in paraosseous tissues develop, that can affect the bone tissue.Thus, the research of A.S. Dmitrenko is devoted to the study of microvasculature of muscle tissue in rats under hypothermia [15].Hypothermia was modelled by a single maintenance of rats in chamber while decrease of rectal temperature down to 15°C.After 1, 3 and 7 days of hypothermia exposure there were found luminal occlusion of microcirculation vessels and hemocapillaries of muscle tissue due to swelling and expressed destruction of endothelial cells.The authors suggested that the found changes in microvasculature might affect the bone tissue state due to development of hypoxia in it, whereas microcirculatory bloodstream of bone maintained the partial pressure of oxygen in tissue fluid and supplied cells with nutrient and regulatory substances.It is known that bone formation may occur only in the presence of oxygen, i.e. destructive changes in microvasculature of bone are accompanied by disordered differentiation of precursor cells in osteogenic direction, inhibition of bone cell metabolism (osteocytes, osteoblasts) and delay of bone regeneration processes and defect of remodelling.
Hypothermia may be caused not only by environmental cold stress, but also effect of some medicines.When hypothermia (33.5°C) induced in mice by intraperitoneal administration of reserpine for 4 days there was found an increase of micronuclear cells number (nucleus diameter < 1/4 of cytoplasm diameter) in a bone marrow [27].The authors associate these facts with the defect of mitosis mechanisms in cells.The found changes in bone marrow cells can lead to reduction in the number of osteoblast precursor cells that, in turn, results in inhibiting the bone formation.
both in cell culture, and at an organism level it has been found that the mechanism of hypothermia effect is complicated.Hypothermia affects both directly the bone, i.e. impairing structural and metabolic characteristics, changes the microelement composi-tion and mineral density, and indirectly does by triggering systemic disorders in a body: antioxidant system, balance of hormones, blood supply, etc.
Under hypothermia in bone tissue there were revealed significant disorders of ultrastructural organization of osteoblasts, increase of osteoclastogenesis and decrease of osteoblastogenesis promoting activation of bone resorption.Reduction of osteoblastogenesis may be associated with a decrease in a number of bone marrow stromal cells which can differentiate towards osteogenesis and disordered mitosis phases in osteoblasts.Metabolic processes in bone cells are impaired.In osteoblasts the biosynthesis of alkaline phosphatase and osteocalcin, being a part of organic matrix of bone, reduces.The level of membrane glycoproteins responsible for cell adhesion changes, the content of Ca 2+ -ATP-ase decreases, resulting in accumulation of calcium in cells and contributes to their death.A distinct link between osteoprotegrin biosynthesis decrease with cells and increase of osteoclasts' activity has been established.However, the literature does not provide an information on peculiarities of structural changes of compact and spongy bone, remodelling bone matrix, ultrastructural organization of osteocytes and osteoclasts, microvasculature of bone, bone marrow cells.There were no studies about hypo-thermia effect on bone tissue of animals of all the ages and its remodelling.There studies may be useful for preventing the development of osteoporosis in aged people.
Further study of hypothermia effect of different regimens on structural and metabolic indices of bone and revealing the defect mechanisms of the remodelling of compact and spongy bone under these conditions is a prospective research direction.