Physical training and connective tissues in young mice: biochemistry of long bones

The effect of physical training on collagen, ground substance, and nucleic acid concentrations in long bones was studied in male mice of NMRI strain. The mice to be trained and their controls were about 2 wk old at the beginning of training, which took place on a 5 degrees inclined treadmill 5 days/wk for 3-22 wk. The duration of daily exercise was increased progressively over 3 wk. The final daily exercise bouts were 50 and 80 min for moderate programs and 180 min for the intensive program at a speed of 30 cm/s. Increased concentrations of nitrogen and hexosamines were found at both training intensities, especially after prolonged training. No conclusive changes in nucleic acid concentrations were observed after training. The hexosamine-hydroxyproline ratio was higher and the hydroxyproline-nitrogen ratio lower in the long bones of trained animals compared to the controls. In conclusion these data suggest that prolonged physical activity affects the organic matrix of long bones by maintaining above average concentrations of glycosaminoglycans in matured bones.     

Physical training and connective tissues in young mice-heart

The effect of physical training on the chemical properties of the heart tissues was studied in male mice of NMRI-strain. The mice to be trained and their controls were about 2 weeks old at the beginning of the training, which took place on 5 degrees inclined treadmill 5 days a week for 3-22 weeks. The duration of daily exercise was progressively increased over the first 3 weeks. The final daily exercise bouts were 50 and 80 min for moderate programs and 180 min for the intensive program at a speed of 30 cm/s. The whole heart or the ventricles were used for the analyses. We found no significant changes related to training in the concentrations of nitrogen, hexosamines, and hydroxyproline both in the normal-sized and the hypertrophied hearts. The hydroxyproline concentration of the ventricles was lower than that of the whole heart tissue, but no difference was observed in the concentration of nitrogen. The hydroxyproline concentration of the heart tissue increased with age both in the trained and untrained mice (39% over 19 weeks). We conclude that collagen and non-collagen proteins in the heart tissue of young mice are stimulated in equal proportions by physical training.     

Blood circulation of long bones in trained growing rats and mice.

The effect of physical training on the blood circulation of long bones was studied in growing rats and mice of NMRI-strain. The animals to be trained and their controls were about 2 weeks old at the beginning of the training. The training took place on a 5 degree inclined treadmill 5 days a week for 3 weeks in experiment I and 7 weeks in experiments II and III. The duration of the daily exercise was progressively increased over 3 weeks. The final exercise bouts were 80 min for moderate and 180 min for intensive training programs. The circulating red cell volume (ml/100 g bone) of the humeral, femoral and tibial bones of the trained animals was lower compared to the controls in all three experiments mainly due to reduced hematocrit values. The circulating blood volume (ml/100 g bone) decreased in the tibial bones of the trained animals in experiment I and showed a decreasing tendency in experiment III, but no significant differences between the groups were observed in the humeral and femoral bones. Yet, when related to the volume of the bones the circulating blood volume (ml/100 ccm bone) was significantly higher in the femoral bones of the trained animals, while the changes in the humeral bones were negligible (experiment III). The results suggest that the vascularity of long bones is affected by physical training. The varying responses in different bones are perhaps due to the amount of mechanical stress during physical activity.     

Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice

Several alphaviruses, including the Sindbis-group viruses, Ross River virus, O'nyong-nyong virus, and Chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. Mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. S.A.AR86 is a Sindbis-group alphavirus closely related to the arthralgia-associated Ockelbo and Girdwood S.A viruses. Following inoculation with S.A.AR86 derived from a molecular clone, infectious virus was isolated from bone and joint tissue 1 to 6 days postinfection. Studies using either in situ hybridization or S.A.AR86-derived double promoter viruses and replicons expressing green fluorescent protein localized sites of viral replication to the periosteum, tendons, and endosteum within the epiphyses of the long bones adjacent to articular joints. These results demonstrate that alphaviruses associated with arthralgia in humans replicate within bone-associated connective tissue adjacent to articular joints in an adult mouse model.     

Physical training in obese women. Effects of muscle morphology, biochemistry and function

Peripheral adaptations to 3 months of physical endurance training without food restrictions were studied in skeletal muscles of 14, middle-aged, physically untrained, obese women. In comparison to aged-matched controls of normal weight, the obese group showed significantly lower isometric endurance. In the obese group, physical training resulted in a significant increase of maximal isometric and isokinetic strength. Isokinetic but not isometric endurance also increased after training. The isometric strength of obese women showed a positive correlation with the percentage of FTb fibres. The training (50 min/day, 3 days/w) did not result in any change in body weight, body fat, and the number and weight of fat cells. The 20% increase of VO2 max after training was found to be significantly correlated with the increase in the number of capillaries around muscle fibres. The relative percentage of FTa fibres, the number of capillaries per fibre as well as the activities of citrate synthase, 3-hydroxy-acyl-CoA-dehydrogenase, and hexokinase showed a significant increase after training. The concentrations of glucose during OGTT showed a trend to decrease with a significant decrease at the end glucose curve (120-min value). The concentration of insulin and C peptide and the insulin removal did not change after training. The changes in the concentration of glucose during OGTT was significantly correlated with the increase in muscle capillarization and of dynamic endurance.     

The pattern of ATP-creatine phosphotransferase activity in growing long bones of new-born mice

Synopsis The localization of creatine kinase (ATP-creatine phosphotransferase) in growing long bones of new-born mice was studied histochemically. Its activity was found to vary within and between different areas of the cartilagenous epiphyses of proximal tibia. Chondrocytes in the cartilaginous epiphysis showed a high cytoplamatic activity. Active enzyme was also apparent within the growth plate of the diaphysis (with the exception of degenerated cells), in the metaphyseal part of the growth plate within the matrix and in neighbouring areas of the metaphysis, and in areas containing lamella bone. Periosteum and perichondrium also showed signs of enzyme activity. The significance of these findings is discussed in relation to the growth process.     

An in vitro assay of bone development using fetal long bones of mice: morphological studies

The purpose of this study was to examine the morphological changes in an in vitro system in which the two elements of bone modelling, formation and resorption, could be studied simultaneously. Pregnant mice were killed on days 15, 16 and 17 of gestation, the fetuses were removed and the radii and ulnae dissected free of soft tissue. The bones were cultured for 6 days in media (BGJ) supplemented with 20% fetal calf serum and 150 micrograms/ml vitamin C. Growth and mineralization were estimated by measuring the total length of the bone, and diaphysis, and by light and transmission electron microscopy (TEM). The results of this study indicate that there is a continuous measurable increase in the total length of fetal mouse long bones over the 6 days of culture. These bones show a continuous growth of periosteal bone, with mesenchymal tissue penetrating into the diaphyseal shaft, and development of bone marrow like tissue. TEM examination showed differentiation of mesenchymal cells to osteoblasts, formation of new bone matrix and bone mineralization similar to that found in developmentally matched controls. In the cartilagenous epiphyses, however, many hydroxyapatite crystals were not associated with matrix vesicles. In addition, some of the chondrocytes of the hypertrophic zone appeared to be dedifferentiating into mesenchymal cells with osteoblast-like features. In spite of the lack of osteoclasts in the 15- and 16-day explants, osteoclasts appeared in the diaphysis after 2 and 4 days in culture. Our results suggest that this system can serve as a good model for the study of bone formation and resorption as they occur, simultaneously, during bone modelling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemistry of sialo-mucins in connective tissues

Summary The present investigation was undertaken in an effort to differentiate histochemically between sialo-mucins and acid mucopolysaccharides in connective tissues.On the basis of previous studies regarding the histochemical identification of sialic acid in sections of animal salivary glands the author applied different technical procedures on cartilage and tooth germs of newborn rats.The results obtained indicated that in the tissues examined sialic acid is a normal component of the polysaccharide fraction of the connective tissue ground substance.On leave of absence at the University of Rome, Medical School, Viale Regina Elena 287-A, Rome, Italy.     

Congenital bowing of long bones: clinical and experimental study

Offspring of rat dams that consumed isocaloric liquid diets containing either 35% or 0% ethanol-derived calories (EDC) from gestation days 6-20 were tested for play-fighting behavior as juveniles. Offspring from a group of dams maintained on standard lab chow and water throughout gestation were also included. Animals were tested in pairs, with offspring from each of the three prenatal treatment conditions (35% EDC, 0% EDC, and lab chow) being paired with another same-sex animal from one of these three prenatal treatment groups. Although play-fighting in juveniles is normally sexually dimorphic, this normal pattern was absent in juveniles prenatally exposed to alcohol. Male alcohol-exposed offspring displayed feminized behavior while female alcohol-exposed offspring showed masculinized behavior. This reversal of the normal sexually dimorphic aspects of play suggests that some of the behavioral disturbances associated with prenatal alcohol exposure may result, in part, from an alcohol-induced disruption of the hormonal environment in which the fetus develops.     

Functional differentiation of long bones in lorises

The external dimensions of the limb bones and the geometry of their midshaft cross-sections were determined for Loris tardigradus and Nycticebus coucang. Relative cortical thickness, cortical area, and second moment of area were calculated and contrasted with locomotor stresses. The difference in shape-related strength of the bones between the smaller- and the larger-bodied species is more pronounced than can be expected from stresses acting during normal locomotion. The Nycticebus skeleton has a much higher safety margin overall and seems to be dimensioned for infrequent but critical stresses of high magnitude. Lorisine gaits in general are characterized by low ground reaction forces, great mobility in all joints, and a nearly equal share in propulsion and weight-bearing by the fore- and hindlimb. Accordingly, the long bones of lorises (especially those of L. tardigradus) tend to be less rigid than those of other mammalian species (including other primates), they lack a preferential plane of higher bending strength, and femur and humerus do not differ markedly in their capacity to withstand mechanical stresses. External dimensions of the humerus and femur of the two African lorisine species parallel and corroborate these results. Some more general implications for the relationships between bone shape and locomotor stresses are also discussed.     

Oxygen and the connective tissues.

Avascular connective tissues (cartilage, discs, cornea) change with maturation and aging, particularly in large animals, where diffusion paths are longest. It is suggested that the changes in such tissues are responses to increasing difficulties in obtaining oxygen. Two almost identical structural polymers are made in these tissues: chondroitin sulphate, which requires large amounts of oxygen for biosynthesis and keratan sulphate, which requires relatively little. The observed balance of these polymers in the tissue is proposed to depend on the control of biosynthesis by the ambient oxygen tension, and/or selective breakdown.     

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.     

Irradiation-induced hypoxia in bones and soft tissues: an experimental study

Bone marrow and subcutaneous tissue pO2 and pCO2 were measured by means of implanted tissue tonometers in irradiated and nonirradiated rabbit hind limbs. The x-ray dose was 500, 1000, 1500, 2000, and 3000 rads. Tissue gas tensions were measured 1 day and 5 and 11 weeks after radiation. The pCO2 changes in both tissues were slight but not statistically significant. The subcutaneous tissue pO2 decreased during the acute phase of irradiation injury, and the effect of irradiation was dose-dependent. Later on, irradiation had no significant effects on the subcutaneous pO2, although light microscopy of the affected tissues showed fibrosis and blood vessel changes. The response of the subcutaneous pO2 to systemic hyperoxia also increased in the chronic phase of irradiation injury as a sign of improved microcirculation. The bone marrow showed a high radiosensitivity. Irradiation caused a rapid dose-dependent decrease of the marrow pO2, and the marrow pO2 decreased with time during the chronic phase of irradiation injury. The marrow pO2 responded slowly and marginally to an increment of arterial pO2 during breathing 100% oxygen as further evidence of impaired vascular pattern. The results showed that irradiation causes only a transient impairment of tissue perfusion in the skin. However, irradiation-damaged marrow was characterized by progressive tissue hypoxia.     

Synostosis dynamics in human long tubular bones

More than 8,000 roentgenograms and electroroentgenograms of brachial, ulnar, radiocarpal, coxofemoral, knee, talocrural joints have been studied in persons of both sex at the age from birth up to 25 years. The synostosis degree is appreciated by the six-mark system. The roentgenological data are corroborate histologically. The method of appreciation applied makes it possible to reveal cases of initially forming synostosis long before puberty. Three stages of synostosis process are defined. The first stage--slow increase of the synostosis mark. The second--quick synostosis formation. The third--final stage. According to the stages defined, a comparative analysis of the synostosis process in all metaepiphyseal zones of all long tubular bones, peculiarities of the process depending on the sex are demonstrated. The data on the time, when the points of ossification appear, on the beginning and completion of synostosis process in all metaepiphyseal zones of the long tubular bones are presented.